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Plant Physiology Preview. Published on September 6, 2011, as DOI:10.1104/pp.111.183160
Multiple lines of evidence localise signalling, morphology and lipid biosynthesis
machinery to the mitochondrial outer membrane of Arabidopsis thaliana
Owen Duncan1, Nicolas L. Taylor1,2, Chris Carrie1, Holger Eubel1, Szymon KubiszewskiJakubiak1, Botao Zhang1, Reena Narsai1,3 A. Harvey Millar1,2 and James Whelan1*
1
Australian Research Council Centre of Excellence in Plant Energy Biology, 2Centre for
Comparative Analysis of Biomolecular Networks (CABiN), 3Centre for Computational
Systems Biology, M316, The University of Western Australia, 35 Stirling Highway, Crawley
WA 6009, Western Australia, Australia.
* Author to whom all correspondence is to be addressed
James Whelan
ARC Centre of Excellence in Plant Energy Biology
MCS Building M316
University of Western Australia,
35 Stirling Highway,
Crawley 6009,
Western Australia, Australia
Fax: 61-8-64884401
Email: [email protected]
The author responsible for distribution of materials integral to the findings presented in this
article in accordance with the policy described in the Instructions for Authors is James
Whelan ([email protected])
Running title: Arabidopsis mitochondrial outer membrane
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Copyright 2011 by the American Society of Plant Biologists
Abstract
The composition of the mitochondrial outer membrane is notoriously difficult to
deduce by orthology to other organisms and biochemical enrichments are inevitably
contaminated with the closely associated inner mitochondrial membrane and endoplasmic
reticulum. In order to identify novel proteins of the outer mitochondrial membrane we
integrated a quantitative mass spectrometry analysis of highly enriched and pre fractionated
samples with a number of confirmatory biochemical and cell biology approaches. This
approach identified 42 proteins, 27 of which were novel, more than doubling the number of
confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for
the plant outer mitochondrial membrane. The novel components identified included proteins
which affected mitochondrial morphology and / or segregation, a protein which suggests the
presence of bacterial type Lipid A in the outer membrane, highly stress inducible proteins, as
well as proteins necessary for embryo development and several of unknown function.
Additionally, proteins previously inferred via orthology to be present in other compartments,
such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acids accumulation in
developing seeds, were shown to be located in the outer membrane. These results also
revealed novel proteins, which may have evolved to fulfil plant specific requirements of the
mitochondrial outer membrane and provide a basis for the future functional characterisation
of these proteins in the context of mitochondrial intracellular interaction.
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Introduction
Mitochondria are double membrane bound organelles. Whilst the inner membrane
and its role in oxidative phosphorylation has been extensively studied (Eubel et al., 2004),
the outer membrane is often overlooked and has been only lightly studied in plants. Much of
what is known about the functions of the outer membrane has been inferred from studies
carried out in yeast (Saccharomyces cerevisiae) and human (Homo sapiens). Whilst this
approach has been successful in determining the molecular identities of several key
functions of the mitochondrial outer membrane - such as the protein import pore translocase
of outer membrane 40kDa subunit (TOM40) (Jansch et al., 1998) and the regulators of
mitochondrial distribution and morphology - mitochondrial Rho type GTPases (MIRO)
(Yamaoka and Leaver, 2008), lineage specific evolution and specialization of mitochondrial
function has limited the applicability of much of the data gathered in other species. For
example, only two of the six components making up the translocase of the outer membrane
(TOM) complex in plants; TOM40 and TOM7, have been identified in plant genomes on the
basis of sequence identity (Werhahn et al., 2001). Also, outer mitochondrial membrane
proteins, such as OM64, which exhibits sequence similarity to a well characterized plastid
localized protein, would be missed or incorrectly annotated, in an orthology based approach
comparing mitochondrial proteins from other organisms (Chew et al., 2004).
Using green fluorescent protein tagging (GFP), mitochondria have been visualised to
undergo fusion, fission and rapid movements, suggesting a dynamic interaction with
components of the cytoskeleton (Sheahan et al., 2004; Sheahan et al., 2005; Logan, 2010).
However, many of the specific proteins that mediate such processes remain unknown. While
mitochondria do play a central role in programmed cell death in plants, it is clear that,
compared to mammalian systems, different protein components mediate this process. For
example, plant genomes do not appear to have genes encoding the B-cell lymphoma 2 (Bcl2) family of proteins, which are known to mediate mitochondrial membrane permeability
shifts (Reape and McCabe, 2010). The outer membrane not only acts as a barrier for
molecules from the cytosol to enter mitochondria, it also acts as a barrier for molecules to
leave mitochondria. Thus, the outer mitochondrial membrane may also contain proteins
involved in signal transduction and mediating retrograde signals from the mitochondrion to
the nucleus or even from the mitochondrion to the plastid. Proteins such as nuclear control
of ATPase (NCA-2) (Camougrand et al., 1995) in yeast and the mammalian mitochondrial
antiviral signalling protein (MAVS) (Koshiba et al., 2011) are examples of such outer
mitochondrial membrane signalling components identified in other species, none of which
have thus far been localized in plants.
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The identification of proteins localized to the mitochondrial outer membrane is
complicated by its relatively low protein content and its close association with the protein rich
inner membrane and endoplasmic reticulum. Furthermore, in plants, it has been shown that
the inner and outer mitochondrial membranes are physically linked by a c-terminal extension
of the translocase of the inner membrane protein, TIM17-2, which is anchored in both
membranes (Murcha et al., 2005). Similarly, in yeast, the Mitochondrial Distribution and
Morphology protein, MDM10 (an ortholog of which has not been found in plant genomes)
has been shown to link the mitochondrial and endoplasmic reticulum membranes (Kornmann
and Walter, 2010). Attempts to isolate pure outer membrane fractions are therefore likely to
be compromised by contamination from either of these two structures. Previous studies
aimed at determining the outer membrane proteome by direct analysis of such fractions
have often suffered from either limited coverage (30 proteins found, 67 % estimated
coverage in Neurospora crassa; (Schmitt et al., 2006)) or an excess of contaminants (112
proteins found, many likely contaminants, estimated 85 % coverage in Saccharomyces
cerevisiae; (Zahedi et al., 2006)). Coverage estimates are calculated by summing the total
numbers of proteins previously localized to the outer membrane and determining what
percentage of these are independently identified in these studies. In the case of Arabidopsis
and plants in general, obtaining pure outer mitochondrial membrane fractions is further
complicated by the presence of the chloroplast, and a greater difficulty in obtaining sufficient
quantities of mitochondria necessary to perform sub-organelle proteomics on low abundance
fractions.
With these challenges in mind, we developed a statistically rigorous quantitative
proteomic workflow to provide a qualitative assessment of sub-organelle protein location in
Arabidopsis. We then coupled this with independent evaluation by biochemical and cell
biology approaches (Millar et al., 2009), in order to confidently determine components of the
outer mitochondrial membrane proteome of Arabidopsis. Firstly, we enriched outer
membrane vesicles from highly purified mitochondria and compared the abundance of its
constituents to that in pre-fractionated ‘contaminant’ samples. The proteins found to be
enriched in the outer membrane fractions were confirmed or discarded as bona fide outer
membrane proteins based on investigation through the literature and further experimental
confirmation by GFP tagging and transient expression, or by in vitro import of
35
S-Met
labelled precursor proteins into purified mitochondria. By hierarchical evaluation of this data,
we identified 42 mitochondrial outer membrane proteins, 27 of which are novel to this
localization for an estimated 88% coverage. The proteins identified range from plant specific
proteins with unknown functions to proteins that have putative functions in mitochondrial
signalling, morphology and defence responses.
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Results
In order to identify protein components of the mitochondrial outer membrane we
developed a workflow that used multiple lines of independent evidence to discover and
confirm the sub-cellular localization of proteins on the mitochondrial outer membrane. The
first aspect of this workflow was the selection of multiple, independent pre-fractionated
samples for comparisons with enriched mitochondrial outer membranes in order to
independently analyze the likely causes of contaminants (Figure 1A). Furthermore, we
sought a control for the protein composition of other mitochondrial membranes (IM), a
control for co-enrichment of contaminating proteineous structures in the outer membrane
isolation process (HSP-OM) and a control for non-membrane proteins associating with the
outer membrane during the cell fraction procedures (Mitoplasts). By comparing the
abundance of the proteins present in each of these fractions (Figure 1B) we were able, in
effect, to subtract contaminants from the mitochondrial outer membrane fraction in a
statistically rigorous fashion (Figure 1C). It should be noted that the success or failure of this
technique therefore, hinges not on the purity of the fractions analyzed but on the relative
enrichment of sources of contamination in the pre-fractionated samples to levels greater
than that observed in the mitochondrial outer membrane fraction. The result of these
analyses was termed the putative outer mitochondrial membrane proteome. The putative
sub-cellular / sub-mitochondrial location for each of these proteins was then assessed by
techniques independent of the fractionation procedure, including green fluorescent protein
tagging or in vitro mitochondrial protein import experiments. To rigorously define the outer
mitochondrial membrane proteome, candidate proteins needed two independent pieces of
evidence, such as enrichment in the outer membrane fraction as determined by mass
spectrometry, and, confirmation by another independent approach such as in vitro or in vivo
targeting ability to the outer membrane or western blot analysis (Millar et al., 2009). The
application of these criteria lead to the identification of a stringent set of 42 proteins (Figure
1C).
Preparation of mitochondrial outer membrane and pre-fractionated samples
To enrich mitochondria, from which outer membranes could be isolated, a two-stage
separation of the organelle fraction of Arabidopsis protoplasts was carried out. This involved
first obtaining a crude organelle fraction (referred to as high speed pellet, HSP) and then
enriching mitochondria from this fraction, initially by density gradient centrifugation and
subsequently by free flow electrophoresis. The ability of free flow electrophoresis
fractionation to separate mitochondria from other cellular contaminants, even those in close
association with the mitochondria (such as the endoplasmic reticulum) on a basis
independent of organelle density (i.e. surface charge) allows mitochondria to be enriched to
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a greater degree than single physical property isolation strategies (Figure 2). The
mitochondrial enrichment of each of these fractions was assessed by comparison of
Coomassie blue staining profiles (Figure 2A) and immuno-detection (Figure 2B & C) of
proteins found in mitochondria (40 kDa subunit translocase of outer membrane - TOM40),
peroxisomes (3-Ketoacyl-CoA thiolase - KAT2), plastids (ribulose bisphosphate carboxylase,
large subunit – RbcL and Light harvesting complex B - LhcB), and the endoplasmic reticulum
(CALNEXIN) (Figure 2). Coomassie blue staining of proteins separated by SDS-PAGE
indicated that both the gradient fractionated and free flow electrophoresis (FFE) fractionated
samples were enriched in mitochondria compared to the starting material - the high speed
pellet (HSP) (Figure 2A). Image based quantitation of band intensity following immunodetection confirmed this enrichment as indicated by the presence of the outer membrane
marker, TOM40-1, which was 2.5-fold higher following gradient fractionation and 3-fold after
FFE (Figure 2B). Levels of endoplasmic reticulum were assessed with an antibody raised to
recombinant human CALRETICULIN (which primarily detects the membrane bound form of
this protein, CALNEXIN in Arabidopsis as evidenced by size and solubility of the detected
protein). These images indicated that endoplasmic reticulum levels were reduced 14-fold by
gradient fractionation and more than 400-fold by the FFE process. Two major bands were
detected in the HSP fraction (Figure 2B) corresponding to the two CALNEXIN isoforms
AT5G07340.1 (61.4 kDa) and AT5G61790 (60.4 kDa). Peroxisomal contamination, as
indicated by the presence of KAT2 was reduced by 1.5-fold after gradient fractionation and
28-fold following FFE enrichment. Similarly, antibodies raised to ribulose 1, 5-bisphosphate
carboxylase/oxygenase large subunit (LSU rubisco) showed a 4-fold reduction in chloroplast
contamination after gradient purification and a 60-fold reduction following FFE (Figure 2B).
An additional membrane bound plastid marker, Light harvesting complex B – LhcB was
readily detectable in enriched plastid samples and present in the HSP fraction but was
undetectable in either of the mitochondrial enriched fractions (Figure 2C). This high level of
enrichment laid a firm foundation for the subsequent identification of extra mitochondrial
contaminants present in outer membrane fractions.
Mitochondria were then sub-fractionated in order to separate the inner and outer
membranes. This process is perhaps the most problematic step in the enrichment of
mitochondrial outer membrane as the inner and outer membranes are physically linked by
proteins such as TIM17-2 (Murcha et al., 2005). In order to separate them, mitochondria
were placed in hypo-osmotic solution, swelling the highly folded inner membrane to beyond
the capacity of the unfolded outer membrane. This ruptures the outer membrane which is
then dissociated from the inner membrane by several gentle strokes with a Potter-Elvenhjem
homogenizer. The protein rich, dense inner membranes are separated from the less dense
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outer membranes by sedimentation and subsequent flotation through sucrose gradients.
This membrane fractionation was performed on two samples, the first being FFE enriched
mitochondria to yield enriched mitochondrial outer membrane (Mt OM), the second being the
crude organelle enrichment HSP to yield a contaminated outer membrane fraction (HSPOM). Additional pre-fractionated samples consisting predominantly of mitochondrial inner
membranes (Mt IM) and mitochondrial inner membranes and matrix (Mitoplast) were
prepared to allow the identification of contaminants arising from these compartments. The
composition of these samples was examined by immuno-detection of proteins characteristic
of the ER, plastids and the mitochondrial inner membrane (Figure 3). Coomassie brilliant
blue staining of the separated fractions revealed differing protein profiles for each of the
fractionated samples (Figure 3A). Identification of several high abundance bands in this gel
by in ESI MS/MS showed that the most abundant band in the mitochondrial outer membrane
fraction consists of the previously identified mitochondrial outer membrane proteins –
VDACs 1,2,3 & 4. Identification of another high abundance band present in the prefractionated samples but absent from the outer membrane fraction showed it to
predominantly consist of a subunit of the β-subunit of ATP synthase which is located on the
inner mitochondrial membrane. Details of these identifications can be found in
Supplementary Table VII. Immuno-detection of the mitochondrial outer membrane marker
TOM40 (At3g20000.1) in these same fractions (Figure 3B) revealed strong enrichment of the
outer membrane and corresponding depletion in the pre-fractionated samples. Detection of
TOM40 in the mitoplast and inner membrane samples is a result of the incomplete
separation of outer membrane from the inner during the homogenization process.
Conversely, detection of the mitochondrial matrix marker pyruvate dehydrogenase subunit
E1 (PDH-E1, At1g01090.1), the mitochondrial inner membrane marker cytochrome c
oxidase subunit 2 (COXII, AtMg00160.1), the endoplasmic reticulum membrane marker
CALNEXIN and the soluble peroxisome marker KAT2 revealed depletion of each of these
contaminants in the outer membrane fraction and enrichment in at least one of the prefractionated samples. Detection of KAT2 in the mitoplast sample is a result of residual
peroxisomal contamination of the mitochondrial enrichment procedure and can also be
observed in Figure 2B. Owing to the enriched / unenriched sampling strategy used, this
residual contamination is accounted for by the inclusion of HSP-OM which is more enriched
in peroxisomes and plastids than Mt-OM (Figure 2B), however KAT2, being a soluble protein
is lost in the preparation of this membrane sample. Immuno-detection of additional
membrane anchored proteins characteristic of the plastid outer envelope (TOC159
At4g02510.1) and peroxisomal membrane (PMP22, At4g04470.1) did not detect these
proteins in any of the sub-fractionated samples.
7 However the chloroplast membrane
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proteins TOC159 (data not shown) and LhcB (Figure 2C) were detected in purified
chloroplasts.
Mass spectrometry analysis of outer membrane composition
The first quantitative approach undertaken involved the counting of spectra that
matched to individual proteins in the purified outer membrane fraction. These counts were
then compared to those observed in the pre-fractionated samples. The first of these
comparisons was designed to eliminate membrane-associated contaminants, arising from
other organelles/membrane systems (Mt OM:HSP-OM). The second of these comparisons
was designed to identify contaminants in the Mt OM fraction that arose from other
mitochondrial compartments (Mt OM:Mt IM). These comparisons enabled a quantitative
assessment of whether their constituent proteins were located on the outer mitochondrial
membrane or elsewhere in the cell, allowing the assembly of a short-list of likely outer
membrane proteins.
Each of the protein samples (MtOM, MtIM and HSP-OM) were acetone precipitated,
digested and analyzed by ESI-MS/MS. A total of 751 unique Arabidopsis proteins were
identified across three biological replicates of this set of samples. A false discovery rate for
this data set was empirically determined by compiling the mass spectra from each of the
ESI-MS/MS analyses and searching this file against a decoy (shuffled TAIR 9 dataset)
database. These searches indicated a false discovery rate of 1.5% at the peptide level. Of
the 751 proteins, 185 were identified in the outer membrane fraction (Mt OM) in more than
one biological replicate (Supplementary Table I).
By modeling the numbers of spectra observed for each protein using Poisson
regression, we found a total of 64 proteins that were enriched in the outer membrane
fractions using the following criteria: must be detected in 2 or more biological replicates and
have a p-value of ≤ 0.13 for enrichment over both the HSP-OM and Mt IM samples when the
spectra from all biological replicates are pooled (Supplementary Table II). This p-value was
intentionally less stringent at this preliminary stage in order to maximize the ability of these
analyses to discover putative mitochondrial outer membrane proteins, given that any
unintentionally included contaminants would be removed in the subsequent experimental
confirmation steps. The validity of this analysis was also investigated by green fluorescent
protein (GFP) targeting studies of several proteins falling on either side of this cut-off, which
indicated that loosening the p-value further would increase the number of contaminants
without including more outer membrane proteins in the preliminary list (data not shown).
Proteins that were only detected in the Mt OM samples, provided they were present in more
than one biological replicate, were also included on the basis that these were likely to be
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lower abundance mitochondrial outer membrane proteins, only detected following
enrichment.
This list of 64 proteins did not contain abundant mitochondrial inner membrane
proteins such as the ADP/ATP carrier and was generally devoid of components of the
mitochondrial respiratory chain. The 64 proteins encompassed 15 of the 17 proteins that
have previously been shown to be located in the outer mitochondrial membrane in various
studies in plants (Werhahn et al., 2001; Chew et al., 2004; Scott et al., 2006; Lister et al.,
2007; Xu et al., 2008; Yamaoka and Leaver, 2008; Lee et al., 2009). Despite the difficulties
inherent in working with plant cells, such as limited starting material, lysis complications due
to the cell wall and extra contamination arising from an additional organelle, the percentage
of proteins in this preliminary list compares favorably with previously published outer
mitochondrial membrane proteomes from non-plant species. We calculated 88 % coverage
of known outer membrane mitochondrial proteins in our preliminary dataset vs. 85 % in
Saccharomyces cerevisiae (Zahedi et al., 2006) and 67 % in Neurospora crassa (Schmitt et
al., 2006). This coverage was achieved with a pool of just 64 proteins vs. 118 in
Saccharomyces cerevisiae. The two proteins that were not included in this preliminary set
were TOM7 (At5g41685.1) and BIGYIN (At3g57090.1) (Arabidopsis ortholog of Fis1 in
Saccharomyces cerevisiae and mammals) (Scott et al., 2006).
Literature, mass spectrometry, GFP fluorescence and in vitro import to confirm outer
membrane composition
Additional lines of evidence were sought until each protein present in the 64 member
putative outer membrane proteome had two independent lines of evidence to either confirm
this location or a single high quality line of evidence to dispute its inclusion in this list. A
summary of this information can be found in Table 1 and 2, Supplementary Table III.
Literature
Components of the TOM complex have been previously isolated from mitochondrial
outer membrane preparations and identified by a variety of methods (Werhahn et al., 2001;
Braun et al., 2003), contributing literature evidence for the TOM components: TOM 40-1, 202, 20-3, 20-4, 9-1, 9-2, 5, 6. Other members of the mitochondrial protein import apparatus,
METAXIN (At2g19080.1) and OM64 (At5g09420.1) have previously been shown to be
located in the outer mitochondrial membrane by GFP studies (Chew et al., 2004; Lister et al.,
2007). Several members of the VDAC family, DGS1 (At5g12290.1) and ELM1 (At5g22350.1)
have also been shown to be located in the mitochondrial outer membrane by GFP studies
(Arimura et al., 2008; Xu et al., 2008; Lee et al., 2009) (Table 1).
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A number of proteins present in the putative outer mitochondrial membrane proteome
in this study have previously been reported be to in other sub-cellular locations. These
proteins included TIM9 (At3g46560.1) and TIM13 (At1g61570.1) that have been previously
reported to be soluble proteins of the mitochondrial inter membrane space (Koehler, 2004;
Lister et al., 2005). A variety of other proteins in the set of 64 are known to be proteins in the
inner mitochondrial membrane, notably: Ubiquinone cytochrome c oxidoreductase-like family
protein (At3g52730.1) is a subunit of complex III (Meyer et al., 2008), F0-ATPase subunit 9
(AtMg01080.1) has been shown to be a member of Complex V and FRO1 (At5g67590.1),
unknown protein (At1g68680.1) and unknown protein (At4g16450.1) are subunits of
Complex I (Meyer et al., 2008). IDH1 (At4g35260.1) has also been shown to be present in
the inner membrane/matrix fraction of mitochondria (Zhao and McAlister-Henn, 1996) (Table
2).
Mass spectrometry - iTRAQ
Initial inspection of the putative proteome indicated the presence of soluble
contaminants arising from the mitochondrial matrix. Isocitrate dehydrogenase (IDH6
At3g09810.1) and aspartate aminotransferase (ASP1 At2g30970.1) (Table 2) are two high
abundance matrix enzymes. We chose to address this source of contamination by
conducting an additional quantitative proteomic study using isobaric tags. This study
evaluated the abundance of proteins in four fractions – the same three as used in the
spectral counting analysis with the addition of a sample comprised chiefly of mitoplasts, i.e.
mitochondrial matrix and inner membrane structures depleted in outer membrane. A ratio of
0.7 at p<0.05 was selected as the cut-off on the basis that known outer membrane proteins
were included, whilst known contaminants were excluded. The validity of this cut-off was
confirmed by GFP localization of IDH6 (At3g09810.1, iTRAQ mitoplast / outer membrane
ratio 0.8, geometric standard deviation 1.176) and GABA aminotransferase (At3g22200.1,
iTRAQ mitoplast / outer membrane ratio 0.776, geometric standard deviation 1.185), two
proteins which fell close to but outside the cut-off and were shown to localize to the
mitochondria but not to the outer membrane (Supplementary Figure 3 panels I, II). Thus, the
iTRAQ analysis using these criteria contributed evidence for the removal of 4 proteins from
the list of 64, namely aspartate amino transferase (ASP1, At2g30970.1), Isocitrate
dehydrogenase (IDH6, At3g09810.1), gamma-aminobutyric acid aminotransferase (GABA
aminotransferase, At3g22200.1) and a putative nicotinamide adenine dinucleotidecytochrome B5 reductase (NADH-cytochrome B5 reductase, At5g20080.1).
GFP tagging
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These exclusions left a group of 38 proteins for which additional evidence was
sought in the form of GFP localization. To accurately use this method, it was first established
that GFP and fluorescence microscopy were capable of distinguishing mitochondrial inner
membrane or matrix proteins from those located on the outer mitochondrial membrane
(Figure 4). For these purposes, an inner membrane protein, frostbite1 (FRO1, At5g67590.1) (Heazlewood et al., 2003), a matrix protein - isocitrate dehydrogenase 6 (IDH6,
At3g09810.1) (Heazlewood et al., 2004), and a previously characterized outer membrane
protein elongated mitochondria 1 (ELM1, At5g22350.1) (Arimura et al., 2008) fused to GFP
were transiently transformed into Arabidopsis cell culture along with a mitochondrial matrix
targeted red fluorescent protein control and observed using fluorescence microscopy (Figure
4). A clear difference was observed between the fluorescence patterns of the control
proteins located in the inner membrane – FRO1 (Figure 4A) and matrix – IDH6 (Figure 4B)
and the outer membrane - ELM1 (Figure 4C). The inner membrane and matrix located
controls showed filled, green, circular fluorescence patterns whereas the outer membrane
control (ELM1) displayed green ring patterns. This ring pattern can also be observed for the
unknown function At5g55610.1 (Figure 4D), which was in the set of 38 and deemed to be a
positive localization of the GFP fusion protein to the mitochondrial outer membrane. This is
also consistent with previous reports using GFP tagging of mitochondrial outer membrane
proteins (Setoguchi et al., 2006; Lister et al., 2007). Similar ring structures corresponding to
a mitochondrial outer membrane location were observed for 25 of the 38 proteins tested. Of
these 25 proteins, 21 only displayed this pattern surrounding the red fluorescent protein
(RFP) control (Supplementary Figure 1) indicating an exclusive mitochondrial outer
membrane localization, whilst 4 of the tested proteins displayed fluorescence patterns
consistent with localization to the mitochondrial outer membrane but also localized to other
intracellular structures, which were apparently not mitochondrial in nature (Supplementary
Figure 2). GFP analysis of the remaining 12 proteins showed that 7 of these did not localize
to the mitochondria (Supplementary Figure 4), 2 were seen to be mitochondrial but did not
display the ring structure characteristic of outer membrane proteins (AT5G15640.1,
AT4G04180.1) (Supplementary Figure 3, panel III and IV) and 3 proteins failed to display
any GFP related fluorescence or were inconsistent from cell to cell. Images of all of the
above fluorescence patterns can be viewed in Supplementary Figures 1-4.
In vitro uptake assays
The two mitochondrial localized proteins that did not display the ring structure
(AT5G15640.1, AT4G04180.1) were selected for in vitro protein uptake experiments. Both
were seen to be protease protected when incubated with mitochondria under conditions that
support protein uptake (Supplementary Figure 3, panels III and IV). When valinomycin was
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added to the in vitro uptake assay, no protease protection was evident, indicating the
requirement of membrane potential for import that is a feature of inner membrane or matrix
localizations (Supplementary Figure 3, panels III and IV).
Of the three proteins for which a consistent GFP localization could not be confirmed,
two were deemed to be outer membrane proteins on the basis that other isoforms of the
protein family had been investigated as part of this study and were found to be localized to
the outer mitochondrial membrane. These proteins were SAM50-1 (At3g11070.1) and
MIRO1 (At5g27540.1). The final protein, the unknown protein (At4g17140.2) was excluded,
as no additional evidence was found for its localization beyond the quantitative MS data. The
N and C-terminal 200 amino acids of this large (471.2 kDa) protein were fused to GFP, but
failed to show a consistent sub-cellular localization. Together, the MS, literature searches
and GFP analysis produced a final list of 42 plant outer membrane proteins, 27 of these
being novel, doubling the number of confirmed outer membrane proteins in plant
mitochondria and identifying all but two proteins that were previously known.
Additional confirmation of novel mitochondrial outer membrane localizations
In order to gain further localization confirmation for the three novel mitochondrial
outer membrane proteins: KDSB - At1g53000.1, PECT1 - At2g38670.1 and sorting and
assembly machinery protein SAM50 - At3g11070.1, polyclonal antibodies were raised
following bacterial expression and purification of portions of these proteins. Isolated
mitochondria were treated with increasing amounts of proteinase K. This treatment degrades
portions of mitochondrial proteins that were accessible to the protease and preserved
proteins which were protected by the lipid bilayer of the mitochondrial outer membrane.
Treated mitochondria were separated by SDS-PAGE and a number of proteins were
immuno-detected. TOM20-3 is a mitochondrial outer membrane proteins that is exposed to
the cytosol (Heins and Schmitz, 1996) (Figure 5A). This protein is rapidly degraded at low
concentrations of proteinase K. Examination of KDSB (Figure 5B) revealed a similar breakdown profile indicating that part of this protein is exposed to the cytosol in vivo and is likely to
be located on the outer mitochondrial membrane. PECT1 (Figure 5C) was also seen to be
accessible to the proteinase. Orthologs of Arabidopsis SAM50 have been observed to be
localized to the outer mitochondrial membrane in Saccharomyces cerevisiae (Kozjak et al.,
2003). SAM50, like TOM40 is a trans membrane β -barrel protein descended from the
bacterial ancestor protein OMP85 (Kozjak et al., 2003), and it appears that these two
proteins are not readily accessible to the externally applied proteinase (Figure 5D and E). At
the two highest concentrations of proteinase, both of these proteins were seen to be partially
broken-down, whereas the Rieske iron sulfur protein (RISP) (Figure 5F), which is located on
the inner mitochondrial membrane, was resistant to digestion. This indicates that SAM50, in
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agreement with the other data presented here, is likely to be located on the outer
mitochondrial membrane in Arabidopsis and that mitochondrial integrity was not
compromised by the proteinase treatment.
Transient
over-expression
morphology and segregation
of
outer
membrane
protein
affects
mitochondrial
An unexpected outcome of using full length coding
sequences fused to GFP was the observation that several of the GFP fusion proteins
apparently altered the mitochondrial morphology in transformed cells. In the case of 3deoxy-manno-octulosonate cytidylyltransferase (KDSB - At1g53000.1) highly abnormal giant
mitochondria occupied much of the available space in observed cells (Figure 6A). This
protein appears to be of bacterial origin (Misaki et al., 2009), is thought to be involved in the
synthesis of the outer envelope lipopolysaccharide KDO2-LipidA and has been located in
mitochondria, but not to the outer membrane in previous studies in Arabidopsis (Heazlewood
et al., 2004). Phosphorylethanolamine cytidylyltransferase is the penultimate enzyme
involved in the biosynthesis of phosphatidylethanolamine, an important membrane
phospholipid. The overexpression of the outer membrane targeted fusion protein (Figure 6B)
apparently causes mitochondria to bunch together at one pole of the cell. Overexpression of
a mitochondrial outer membrane NADH cytochrome B5 reductase (Figure 6C) also appears
to alter mitochondrial morphology when compared to the more typical mitochondrial outer
membrane fluorescence pattern seen with the unknown protein At5g55610.1 (Figure 6D).
Transcriptomic analysis of genes encoding outer membrane proteins reveals patterns
of co-expression
In order to determine whether there was a relationship between co-localization and
co-expression, the relative expression levels for the 38 genes that were present on the
Affymetrix ATH1 microarray were analyzed during germination and across the Arabidopsis
developmental microarray series (Schmid et al., 2005). Additionally the patterns of transcript
abundance for 247 genes that encode inner membrane proteins were analyzed to determine
if the transcript abundance of genes that encode outer membrane proteins, or sub-sets of
outer membrane proteins, differed to the patterns of transcript abundance for genes that
encode inner membrane proteins. Data were normalized to make them comparable
(materials and methods) and expression levels were displayed relative to maximum
expression (Figure 7, Supplementary Figure 5).
Overall it was observed that transcript
abundance of genes encoding outer and inner membrane mitochondrial proteins displayed
maximum levels during seed germination, with a subset showing relatively high levels in root
tissues (Supplementary Figure 5). When all genes encoding IM and OM localised proteins
were clustered together over a germination time-course, it was seen that genes encoding
OM proteins were significantly under-represented in Cluster 2 (p<0.05) compared to genes
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
encoding IM proteins, (13% OM v 27% IM) (Figure 7). Gene in cluster 2 display high
transcript abundance in fresh harvested seeds that decrease during stratification. In contrast
45% of genes encoding OM proteins (v 21% of IM protein encoding genes) was seen in the
group of genes showing transient expression during germination in cluster 4 (Figure 7 boxed
in yellow), which represents a significant over-representation (p=0.0007) of genes encoding
outer membrane proteins in this cluster. Genes in cluster 4 peak in transcript abundance
during the first 24 h of germination, and decrease in abundance in young seedlings, as
under these conditions germination is completed at 24 h in Arabidopsis as the radical has
emerged (Weitbrecht et al., 2011). Thus although genes encoding mitochondrial outer
membrane proteins follow the general pattern of that observed for genes encoding inner
membrane proteins in that they display maximum levels of transcript abundance during seed
germination, detailed time course analysis reveals that for a large sub-set (i.e. cluster 4) this
differs to the majority of genes encoding mitochondrial proteins. Previously it has been
shown for maize and rice that active mitochondrial biogenesis precedes the expression of
respiratory chain components of the inner membrane (Logan et al., 2001; Howell et al.,
2006), and thus the pattern observed here suggests that establishments of the proteins of
the outer membrane is one of the earliest steps to take place in mitochondrial biogenesis.
Discussion
The mitochondrial outer membrane is commonly characterized as a relatively simple
phospholipid bi-layer, broadly permeable to small proteins, ions, nutrients, metabolic
substrates and other products of mitochondria. Although broadly true, this simplification of
the functions carried out by the mitochondrial outer membrane belies its involvement in
complex, co-ordinated, cellular processes such as apoptosis (Lindsay et al., 2011), organelle
division (Kuroiwa, 2010), mitochondrial inheritance (Koshiba et al., 2011), lipid synthesis and
trafficking (Osman et al., 2011) as well as selective interactions with the complex cytosolic
environment in the processes of mitochondrial protein import and biogenesis (Walther and
Rapaport, 2009). The involvement of the mitochondrial outer membrane has often been
incidental to the study of these processes; and the unequivocal assignment of functions or
protein components to the mitochondrial outer membrane is commonly avoided in the
literature, significantly due to the difficulties in gathering sufficient evidence to make a
convincing case for outer membrane localization. Studies such as the one presented here
have several important functions; confirmation of some expected but unproven sub-cellular
localizations, the identification of expected, but as yet uncharacterized protein components
of biological processes, and as the basis for launching the investigation of novel,
uncharacterized protein components – with the potential to lead to the assignment of new
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and unexpected biological functions. The analysis of the mitochondrial outer membrane in
plants is especially interesting because the presence of the chloroplast in plants cells
increases the complexity of the intracellular environment and possibly leads to the observed
sequence divergence across kingdoms (Macasev et al., 2000) in many of the protein
components of the outer membrane involved in mitochondrial protein import (Lister et al.,
2007). By systematically characterizing the mitochondrial outer membrane proteome in
plants, we have identified proteins putatively involved in diverse processes including
signaling, cytoplasmic streaming, protein import, protein degradation and membrane
biosynthesis.
Developing a proteomic strategy for characterizing the outer membrane proteome
Developing a strategy for characterizing the proteome of one membrane from a multicompartmented structure within the cell is challenging due to the multiple and overlapping
potential sources of contamination and the probability that biochemical enrichment
techniques will co-purify multiple structures at the same time. This is a recurring theme in
sub-cellular proteomics, especially in sub-cellular membrane proteomics (Lilley and Dunkley,
2008). Combining methods which evaluate the protein content of enriched membranes
relative to contaminating structures with visual and biochemical assays such as in vitro and
in vivo protein import provide a solid basis for determining sub-cellular and sub-organelle
localization. In combination, this is a highly complex and time consuming process to
undertake for a large range of proteins. However, without it, many researchers are too often
following up unexpected proteomic identifications by using genetic or functional assays,
simply to discover that the false discovery rate of the proteomics was too high in the first
place.
Quantitative proteomic tools like spectral counting and iTRAQ offer powerful,
unbiased analysis of enrichment of known and unknown proteins between samples.
However, we have found that using Exponentially Modified Protein Abundance Index
(emPAI) and Normalized Spectral Abundance Factor (NSAF) for quantitative analysis of
spectral counting (Ishihama et al., 2005; Zybailov et al., 2007) and parametric analysis of
mass tag abundances in iTRAQ spectra (Chong et al., 2006) are not immediately suited to
assessments of enrichment. Typically these processes were developed for analysis of small
changes of protein abundance in a backdrop where most proteins are not changing in
abundance. However, in enrichment studies (including the study presented here), most
proteins are changing in abundance, and significant enrichment leads to the identification of
low abundance proteins which are not detected in more crude samples. These problems
affect statistical analysis of both spectral counting and iTRAQ data as datasets are not
normally distributed in the cases of the most enriched and interesting proteins from the
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perspective of sub-cellular location. Furthermore, while the degree of enrichment might be
interesting in these types of studies, the primary aim is to use quantification to give a
qualitative assessment of cellular location. By using quantitative spectral counting and
iTRAQ data to provide a qualitative assessment of location and coupling this to independent
confirmation data for this qualitative assessment, we have developed a workflow for a
confident assessment of the mitochondrial outer membrane proteome that is likely to be
useful for research on any specific membrane in the cell, particularly for analysis of
membrane systems of low abundance (Figure 1).
There are three key aspects of this workflow. Firstly, selection of multiple
independent pre-fractionated samples for comparisons with the enriched fractions in order to
independently cover likely causes of contaminants; in on our case we sought a control for
other mitochondrial membranes (Mt IM), a control for co-enrichment of contaminating
structures in the sucrose gradients (HSP-OM) and a control for non-membrane proteins
associating with the membrane during the cell fraction procedures (Mitoplasts). Secondly,
inclusion of proteins observed in enriched fractions and absent from pre-fractionated
samples. Such proteins should be included despite issues related to the normality of
distributions and limited spectral counts, as long as spectra are sufficient to prove
identification as these include low abundance proteins only found through enrichment.
Thirdly, multiple confirmation strategies that are independent of the enriched fractions are
very helpful, especially ones that relate to targeting of proteins in situ and/or in vitro to the
location identified. In this study, we used diverse literature of counter-claims, GFP labeling in
intact cells and in vitro protein import. In addition to this, protein-protein interaction, in situ
immunohistochemistry and activity assays could also be included for specific studies (Millar
et al., 2009).
Proteins of the mitochondrial outer membrane proteome
Using this workflow, 42 high-confidence identifications of outer mitochondrial
membrane proteins were made (Table 1). Only 16 of these proteins were previously
experimentally determined to be located in the outer mitochondrial membrane in plants (Werhahn et al., 2001; Chew et al., 2004; Lister et al., 2007; Arimura et al., 2008; Xu et al.,
2008; Lee et al., 2009). These 16 proteins were largely made up of members of the outer
mitochondrial membrane protein import apparatus (TOM complex and associated receptors)
and the voltage dependent anion channels (VDACs), specifically; the five known import
receptors (TOM20-2, 20-3, 20-4, OM64 and METAXIN) and components of the central
import pore (TOM40, 9-1, 9-2, 5, and 6) and VDAC1-4. Two other proteins, DGS-1
(At5g12290.1) (Xu et al., 2008) and ELM1 (At5g22350.1) (Arimura et al., 2008) have also
previously been shown to be present in the outer membrane. Two proteins that were
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
expected, but not detected in this study were TOM7 (Werhahn et al., 2001) and BIGYIN, the
Arabidopsis ortholog to Saccharomyces cerevisiae and Human Fis1 (Scott et al., 2006).
In addition to the 16 known plant outer mitochondrial membrane proteins, 4 other
proteins were also inferred to be localized to the outer mitochondrial membrane in plants,
based on experimental evidence from other species (Fransson et al., 2003; Kozjak et al.,
2003). Two of these proteins are the SAM50s (At3g11070.1, At5g05520.1), which are
evolutionarily conserved across kingdoms and orthologus of these have also been reported
to be mitochondrial outer membrane proteins in N. crassa (Schmitt et al., 2006) as well as S.
cerevisiae (Zahedi et al., 2006). Similarly, the location of the two mitochondrial Rho type
GTPases (MIRO) like proteins, MIRO1 (At5g27540.1) and MIRO2 (At3g63150.1) were also
inferred to be located on the outer mitochondrial membrane, based on orthology with the
Gem1p gene in Saccharomyces cerevisiae (Frederick et al., 2004). Thus, experimentally
confirming the localization of these in the plants for the first time, further supports a vital and
evolutionarily conserved role for the SAM50s and MIROs on the outer mitochondrial
membrane. Evidence supporting this crucial role can be observed in Arabidopsis knock-outs
of MIRO1, which have been shown to have a seed lethal phenotype (Meinke et al., 2008;
Yamaoka and Leaver, 2008), indicating that this gene is essential for seed viability and
normal plant development. In addition to the aforementioned proteins, a total of 27 proteins
were reported as outer mitochondrial membrane proteins in plants, for the first time in this
study. Examination of these revealed the presence of Hexokinase 1 (HXK1, At4g29130.1),
Hexokinase 2 (HXK2, At2g19860.1) and Hexokinase like protein 1 (HXKL-1, At1g50460.1)
on the mitochondrial outer membrane (Table 1). The Arabidopsis hexokinases have long
been reported to be associated with mitochondria, first in 1983 (Dry et al., 1983; DamariWeissler et al., 2007) and more recently with reference to the association of glycolytic
enzymes with mitochondria (Graham et al., 2007). The presence of a putative trans
membrane region of 20 to 25 amino acids, high hydrophobicity at the N terminus
(Supplementary Figure I, panels IV, VII, XVII), the ability to target GFP to mitochondria,
combined with its enrichment in the outer membrane samples (Table 1), and a lack of
detection of other glycolytic enzymes suggests that these proteins are authentic outer
membrane proteins. The location of these HXKs on the outer mitochondrial membrane in
plants may be crucial for a role in the regulation of programmed cell death, with recent
findings in Nicotiana benthamiana implying a role for mitochondrial HXKs in this process
(Kim et al., 2006). Another study has also proposed a role for mitochondrial hexokinases in
generating ADP to support oxidative phosphorylation and minimize the limitation of
respiration by restraints on ATP-synthesis, thereby playing a role in antioxidant defense (da-
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Silva et al., 2004). Nevertheless, it appears that the presence of hexokinases on the outer
mitochondrial membrane positions these proteins ideally for a functional role in signaling.
Similarly, whilst a NADH:cytochrome B5 reductase (At5g17770.1) might be expected
to be in the outer membrane of mitochondria based on the localization of its Saccharomyces
cerevisiae ortholog (Haucke et al., 1997). There are two NADH:cytochrome B5 reductases in
Arabidopsis, thus the identification of this protein on the outer mitochondrial membrane
distinguishes it from other NADH:cytochrome B5 reductase activities in Arabidopsis, such as
the one encoded by At5g20080, which has been shown to be mitochondrial but is likely to
be located on the inner membrane (Heazlewood et al., 2004). Notably, the protein shown to
be on the outer mitochondrial membrane in this study; NADH cytochrome B5 reductase
(At5g17770.1) has previously been reported to be a microsomal enzyme associated with the
endoplasmic reticulum, even though no direct evidence was presented to determine location
(Fukuchi-Mizutani et al., 1999). A recent study has also shown that a mutation in this
NADH:cytochrome B5 reductase (At5g17770.1) significantly reduced the accumulation of
hydroxyl fatty acids in developing seeds by 85%, confirming an essential role for this protein
in lipid metabolism (Kumar et al., 2006). NADH:cytochrome B5 reductases have been shown
to catalyze the transfer of electrons from NADH to two molecules of membrane bound
cytochrome b5 (Strittmatter, 1965). Taken together, these findings suggest that the reactions
catalyzed by both NADH:cytochrome B5 reductases are likely to occur on the mitochondrial
membranes and not the endoplasmic reticulum, as previously assumed.
Novel proteins of the mitochondrial outer membrane
In addition to the proteins outlined above, a number of other proteins were found to
be located in the outer mitochondrial membrane that could not have been predicted by
sequence orthology alone. These proteins can provide a molecular handle for processes that
occur in mitochondria, where, the identification of molecular components has been limited, to
date.
A novel β-barrel protein - One of the proteins identified in the outer membrane proteome was
a novel β-barrel protein (At3g27930.1), bringing the total number of distinct β-barrel proteins
identified in the Arabidopsis mitochondrial outer membrane to four (TOM40, SAM50, VDAC,
At3g27930.1: Table 1). Amino acid sequence alignments comparing this novel protein to
other membrane β -barrel proteins (TOM40, SAM50, VDAC, MDM10, OMP85 and TOC75)
from diverse species revealed that this β-barrel protein is only found in organisms with a
green chloroplast, from algae to various plant species (data not shown). Although the
function of this protein (At3g27930.1) is unknown (as annotated in TAIR10), upon searching
the literature, it was found that this gene has been identified in the list of 437 proteins making
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
up the predicted Arabidopsis N-myristoylome (Boisson et al., 2003). Given that
N-myristoylation is a permanent modification that affects the membrane binding properties of
cytoplasmic proteins, promoting their association with membranes, it was interesting to find
that At3g27930.1 is this list. Taken together with the confirmed outer mitochondrial
membrane localization (Table1), high expression in seed and early germination, (Figure 7)
and the fact that this protein is plant specific (Plant Specific Protein Database; (Gutiérrez et
al., 2004)), it can be speculated that this protein has a crucial and plant specific role in the
interaction between cytoplasmic proteins and the outer mitochondrial membrane, possibly in
signaling or even as an novel import component.
Proteins involved in synthesis of membrane components - Three proteins that were
confirmed to be located to the outer mitochondrial membrane in the course of this study
catalyze key steps in the synthesis of membrane components (At2g01460, At1g53000 and
At2g38670). At2g01460.1 contains two uridine monophosphate / cytidine monophosphate
kinase (UMPK/CMPK) like domains and is predicted to catalyzes the conversion of uridine
monophosphate / cytidine monophosphate to their di-phosphate forms. It has previously
been demonstrated that a nucleoside di-phosphate kinase (At4g11010.1) is present in the
mitochondrial inter membrane space (Sweetlove et al., 2001). Thus, the reaction of these
two enzymes would yield cytidine triphosphate from a cytidine monophosphate substrate
with conversion of ATP to ADP. An additional protein found to be located to the outer
membrane in this study was PECT1 (At2g38670.1) which is a CTP:phosphorylethanolamine
cytidyltransferase
(Mizoi
phosphoethanolamine
et
onto
al.,
2006).
cytidine
This
enzyme
triphosphate
catalyzes
+PPi
to
the
transfer
yield
of
cytidine
diphosphoethanolamine (CDP-ethanolamine). The final enzyme required for production of
phosphatidylethanolamine is CDP-ethanolamine phosphotransferase, the location of which
is not yet characterized in Arabidopsis but is found in the endoplasmic reticulum in
Saccharomyces cerevisiae (Jelsema and Morre, 1978). Interestingly, despite the fact that
PECT1 is an intermediate in this pathway, it has been shown that when this gene is knocked
out in Arabidopsis, a seed lethal phenotype is observed (Mizoi et al., 2006; Meinke et al.,
2008). Thus, it appears that this role for PECT1 on the outer mitochondrial membrane is
essential for seed viability and development in Arabidopsis.
In the course of confirming these proteins as bona fide outer membrane proteins,
full-length coding sequences were fused to the GFP coding sequence and expressed using
the 35S Cauliflower mosaic virus (CaMV) promoter, resulting in transient over-expression of
the experimental protein. Interestingly, in the case of PECT1, this resulted in abnormally tight
bunching of the mitochondria at one pole of the cell as opposed to the regular evenly
distributed morphology (Supplementary Figure 1, panel IX), suggesting that PECT1 is not
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
only essential for catalyzing the reaction yielding CDP-ethanolamine, but that increasing the
abundance of this protein or perhaps the flux of its reaction can lead to abnormal
phospholipid composition of the outer mitochondrial membrane or association of
mitochondria. Also with a proposed role in membrane synthesis, At1g53000.1 encodes 3deoxy-D-manno-2-octulosonic acid (Kdo) transferase, which catalyzes the transfer of Kdo
onto cytidine triphosphate to form CMP-Kdo (Seveno et al., 2010). This protein is
orthologous to the Escherichia coli protein KdsB, which participates in the synthesis of
KDO2Lipid-A, an essential component of the gram negative bacterial cell wall (Opiyo et al.,
2010). Basic local alignment searching showed that orthologous genes to each of the
essential members of the bacterial KDO2Lipid-A biosynthesis pathway are present in the
Arabidopsis genome and are predicted to be targeted to mitochondria (Raetz and Whitfield,
2002; Heazlewood et al., 2007). Taken together with the histochemical evidence for the
presence of Lipid-A in eukaryotic organisms (Armstrong et al., 2006), the evidence that overexpression of this protein produced giant mitochondria (Figure 4, panel A) suggests the
presence of a KDO2Lipid-A or a related compound in the outer membrane of mitochondria.
AAA-Type ATPases –Two related proteins containing ATPases Associated with diverse
cellular Activities (AAA) type ATPase domains were identified on the outer membrane, these
were At3g50930.1 and At3g50940.1, which are 67% identical at the amino acid level and
appear to be the result of a recent duplication in the Arabidopsis genome. Although the
protein encoded by At3g50940 is annotated as encoding a ubiquinol-cytochrome c
reductase (bc1) Synthesis (BCS)-like protein, closer examination reveals that it only contains
the AAA-ATPase domain in common, and lacks the BCS domain. Interestingly, it differs to
the AAA-ATPase proteases that are located on the inner membrane, and displays closer
similarity to another AAA-ATPase, which has been shown to be a calcium-binding protein
whose function is still unclear, but is dual localized to both mitochondria and chloroplasts
(Bussemer et al., 2009). Given that BCS1 was seen to be stably expressed over normal
development (Figure 7), but has been shown to be among the most stress inducible
transcripts encoding mitochondrial proteins (Van Aken et al., 2009), it may have a more
specific role in the repair of proteins following a wide variety of stresses (Xu et al., 2010).
Proteins involved in signaling and catabolic processes – In addition to the stress inducible
AAA-type ATPases discussed above, a number of other proteins identified in this study, may
have significant roles in signaling. Hexokinases and MIRO have been shown to have roles in
signaling, with hexokinase and hexokinase-like proteins shown to have roles in regulating
ROS levels and plant growth respectively (Karve and Moore, 2009; Bolouri-Moghaddam et
al., 2010), whilst MIRO proteins have been observed in regulating mitochondrial transport
and morphology (Yamaoka and Leaver, 2008; Reis et al., 2009). Another protein that may
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
be linked to signaling is a TraB family protein (At1g05270.1), which plays a role in the
conjugative transfer of plasmids in bacteria (An and Clewell, 1994), however the roles in
eukaryotic cells are still unknown. Nevertheless, the identification of this protein implies a
conserved role for this protein, both on bacterial membranes and evidently on the
mitochondrial outer membrane in plants. Another well conserved protein is embryonic factor
1 FAC1 (At2g38280.1), for which the sub-cellular location has long been discussed (Han et
al., 2006), but in this study we report an outer mitochondrial membrane localization for this
protein, on the basis of both proteomic and GFP evidence. Interestingly, like PECT1 and
MIRO1, it has been observed that plants have a seed lethal phenotype when FAC1 is
knocked-out (Xu et al., 2005; Meinke et al., 2008), indicating a vital role for these outer
mitochondrial membrane proteins in Arabidopsis development.
Conclusion:
While we have identified 42 proteins located at the mitochondrial interface with the
cell cytoplasm in plants, there are likely to be some membrane proteins that have not been
identified in this study. Dual or multi-targeted proteins would be excluded by the strategy
undertaken as they would be detected in the contaminant fractions. The cell culture system
used may not have components that may be present in photosynthetic cells, or they may be
present at levels below the level of detection. No additional components of the SAM complex
of the outer membrane were identified that might be expected to be present in comparison to
the SAM complex from yeast. Nevertheless, 27 proteins were experimentally determined to
be located on the outer mitochondrial membrane for the first time in plants and only one of
these (AT5G60730.1) has been consistently predicted to be mitochondrial (Heazlewood et
al., 2007). Of the other 15 proteins, some had counterparts in Saccharomyces cerevisiae or
mammalian systems, and some have been previously shown to be associated with the
mitochondrial outer membrane. However, many can only be identified as on the outer
mitochondrial membrane based on direct experimental analysis, as they are members of
larger families that have a variety of locations in the cell. Several proteins that were identified
had no functional annotations. For these the discovery of outer membrane localization
provides the first functionally related information for the proteins encoded at these loci.
Examination of the full list of proteins identified (Table 1) reveals that plant outer membrane
functions exist beyond their well-established roles in protein and small molecule import.
Importantly, we discovered that plant mitochondrial homologs were not identified for a range
of proteins known to be located in, and to function in the outer membrane in other
organisms. Examples include the B-cell lymphoma 2 (Bcl-2) like proteins, which plays roles
in cell death in mammalian systems (Lindsay et al., 2010), the GTP protein b2 subunit that
plays a role in regulating mitochondrial fusion in mammalian systems (Zhang et al., 2010),
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Mim1 that plays a role in protein import in fungi (Stefan Dimmer and Rapaport, 2010), and a
peripheral-type benzodiazepine receptor that mediates cholesterol transport in mammalian
cells but is located in the secretory pathway in Arabidopsis (Vanhee et al., 2011). Although
we were unable to conclusively rule out the existence of these proteins on the mitochondrial
outer membrane (expression of these proteins may be tissue specific or of low abundance),
it is likely that that at least some of the proteins that we identified with no known functions
may fulfill similar roles. These findings, together with the identification of 5 plant specific
genes (Plant Specific Protein database; (Gutiérrez et al., 2004), including a novel β -barrel
protein (At3g27930.1) and a protein of unknown function (At5g55610.1) suggests that
despite the conserved presence of mitochondria in eukaryotes, several constituents of the
outer mitochondrial membrane have diverged and specialized in different species. 22 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Materials and methods
Purification of mitochondria from Arabidopsis suspension cell culture
Protoplasts were prepared from 6 day old Arabidopsis suspension cell cultures. Typically,
500 g FW of cells were collected by filtration and incubated for 3 h at 21oC shaking 30 rpm in
1 L of 0.4 M mannitol, 3.5 mM MES, pH 5.7 with 0.4 % (w/v) cellulase and 0.05 % (w/v)
pectolyase (Yakult, Japan). Protoplasts were collected by centrifugation 800 x g for 10 min,
resuspended in 500 mL 0.4 M sucrose, 50 mM Tris, 3 mM EDTA, 20 mM cysteine, 0.1 (w/v)
% BSA pH 7.5 HCl and ruptured in a Potter-Elvehjem homogenizer. Cell debris was
removed by centrifugation 2500 x g for 5 min and the organellar fraction (supernatant) was
pelleted 20,000 x g for 20 min. 1/16th of this material was removed and stored at 4 oC for
preparation of the high speed pellet (HSP) and high speed pellet – outer membrane (HSPOM) sample. The remaining organellar fraction was layered onto discontinuous Percoll
gradients consisting of 18 / 25 / 40 % (v/v) Percoll in 0.3 M mannitol, 10 mM TES pH 7.5 and
centrifuged 40,000 x g for 40 min. The 25 / 40 (v/v) % interphase was removed and added to
300 mL 0.3 M sucrose, 10 mM TES, 0.2 % (w/v) BSA pH 7.5 (sucrose wash buffer) and
centrifuged 20,000 x g for 15 min. Subsequently, the supernatant was removed and the
pellet layered onto 40 % (v/v) Percoll in sucrose wash buffer and centrifuged 40,000 x g for
40 min. The gradient purified mitochondria were removed and washed twice in sucrose wash
buffer. 500 µ g of this sample was removed and stored at 4 oC for later analysis. Typically,
100-150 mg of mitochondrial protein (Bradford assay) was recovered.
The gradient purified mitochondria were further purified by free flow electrophoresis based
on a method defined previously with several alterations (Eubel et al., 2007). The gradient
purified mitochondria were first dispersed by passing them back and forth between two 3 mL
syringes through a 10 µm membrane filter (Biorad, Sydney), and then passed through a 5
µm nylon syringe filter prior to introduction to the FFE separation chamber. EDTA was
excluded from the FFE separation medium. FFE purified mitochondria were collected by
centrifugation 20,000 x g for 10 min and washed in sucrose wash medium 20,000 x g for 10
min.
Purification of mitochondrial outer membranes and generation of contaminant
samples
Outer membranes were purified according to a previously published protocol (Werhahn et
al., 2001). In addition to the mitochondrial outer membranes, a ‘contaminant outer
membrane’ sample was generated by treating the high speed pellet collected from the
protoplast disruption step in a similar manner to the FFE purified mitochondria. A second
contaminant sample primarily consisting of mitoplasts was taken from the 32 / 60 % (w/v)
interphase of the first sucrose gradient. An inner membrane fraction was prepared by diluting
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this in 11 mL of sucrose wash medium before freeze / thawing five times and pelleting at
504,000 x g for 1 h in a fixed angle rotor. An additional contaminant sample consisting of
whole mitoplasts removed from the same 32 / 60 % interphase but not subjected to the
freeze / thawing procedure was included in the iTRAQ experiment.
Western blot analysis
10 µg of membrane fractions and 20 µg of organelle fractions per lane were separated by 14
% (w/v) SDS-PAGE. Proteins were transferred onto nitrocellulose membranes. Primary
antibodies were: Translocase of the outer membrane (TOM40-1) (At3G20000.1) at 1:5000 1
h 21 oC and developed according to manufacturer’s instructions (Roche Sydney)(Carrie et
al., 2009). 3-ketoacyl-CoA thiolase (KAT2) antibody (Germain et al., 2001) was used 1:1000,
1 h, 21 oC. Ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit (LSU Rubisco)
and Calreticulin antibodies was obtained from Abcam™ (Sydney) and used 1:500, 18 h, 4
o
C. E1α antibody was obtained from Tom Elthon (University of Nebraska, Lincoln, NE) and
used 1:2000, 1 h, 21 oC. COXII antibody was obtained from Agrisera (Sweden), used
1:5000, 1 h, 21 oC. Antibodies to TOM20-3 (Lister et al., 2007), KDSB, SAM50, PECT1 and
RISP were created by purifying 6*HIS tagged recombinant proteins from E.coli expression
using Profinia based immobilized metal affinity chromatography (Biorad, Sydney). The
purified proteins were used to generate rabbit polyclonal antibodies (IMVS, Adelaide).
Proteinase K titrations
250 ug of isolated mitochondria were resuspended in 0, 2, 8,32 and 64 mg / ml of proteinase
K (Sigma-Aldrich Sydney) in 2.5 ml of 0.4 M sucrose, 50 mM Tris, 3 mM EDTA, 0.1 (w/v) %
BSA pH 7.5 HCl. Samples were incubated on ice for 30 minutes before dilution in 30 ml of
0.4 M sucrose, 50 mM Tris, 3 mM EDTA, 0.1 (w/v) % BSA pH 7.5 HCl containing 1 mmol
Pefabloc SC (Roche Sydney). Samples were centrifuged 20000 x g for 15 minutes,
resuspended in Lamelli sample buffer and separated by SDS-PAGE.
Mass spectrometry
Label free analysis and statistical assessment
50 µg of outer membrane, high speed pellet outer membrane and inner membrane protein
were individually digested overnight with trypsin (10:1), 10 mM ammonium bicarbonate and
20% (v/v) acetonitrile. Insoluble material was removed by centrifugation 20,000 x g, 5 min.
Samples were then dried down in a vacuum centrifuge and analyzed on an Agilent 6510 QTOF mass spectrometer according to a modification of the methods reported in (Eubel et al.,
2008) , as outlined in detail in the supplementary methods section. Spectra are available via
the
ProteomeCommons
Tranche
24 Project
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hash:
3y16ynJWUCAiKGSfZNprN/paRI32nQA9XM0asqZItzPwnf2jEB/zsnOAoSxno14kymUwnJbyl
Kkk4cA2sGdLN52sAQ0AAAAAAAAHqg==
Peptide count data was compiled from three biological replicates each of the outer
membrane, high speed pellet outer membrane and mitochondrial inner membrane samples
as outlined in detail in the supplementary methods file.
iTRAQ labeling, data and statistical analysis
100 µg of 4 samples (outer membrane (MtOM), contaminant outer membrane (HSP-OM),
mitoplast and inner membrane (IM)), each created by pooling three biological replicates and
analysed according a modification of the method reported in (Shingaki-Wells et al., 2011) as
outlined in the supplementary methods section. Quantitation was carried out using an inhouse quantitation method, that carried out protein identifications and quantitation on
isobaric tags of mass 114-117 (4) at the peptide level. Ratios for individual peptide matches
are obtained from peptides meeting the minimum criteria outlined above and are then
combined to determine ratios for proteins hits using a weighted average. The in-house
method of identification and quantitation, outlier removal, minimum number of required
peptides, and definitions of statistical confidence internals are outlined in detail in the
supplementary methods section.
In vitro imports
In vitro import of putative outer membrane proteins into isolated mitochondria were
performed as previously described (Lister et al., 2007). Data are provided in Supplementary
Figure 3 panels II, III and IV.
Green fluorescent protein analysis
Full length coding sequences (with the exception of At4g17140.2) of putative outer
membrane proteins were cloned into the Gateway® cassette cloning system according to the
manufacturer’s instructions (Invitrogen, http://invitrogen.com/). Coding sequences were
transferred into GFP vectors and co-transformed into Arabidopsis suspension cell culture
with an RFP control by particle bombardment as previously outlined (Carrie et al., 2009). Data
are provided in Supplementary Figures 1, 2, 3 and 4.
Arabidopsis microarray analysis
The Arabidopsis AtGenExpress developmental dataset was downloaded as CEL files (EAFMX-9) from the MIAME ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/).
These CEL files in addition to the 30 CEL files that analyzed expression during germination
were imported and quantile normalized together to enable comparability across these arrays
using Partek Genomics Suite version 6.5 (St. Louis, Missouri, USA). Details for each tissue
are shown in Supplementary Table V. Once normalized, expression values were made
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
relative to maximum expression and hierarchically clustered on Euclidean distance using
average linkage clustering. Note that 4 genes; At1g24267, At3g11070, At3g50940 and
At5g22350 were not represented on the ATH1 microarray. This method of normalization and
clustering has been used effectively before to examine co-expression (Narsai et al., 2010).
Abbreviations
COXII
Cytochrome oxidase 2
DGS1
Digalactosyl diacylglycerol deficient suppressor 1
ELM
Elongated mitochondria
FFE
Free flow electrophoresis
GFP
Green fluorescent protein
HSP
High speed pellet
HSP-OM
High speed pellet-outer membrane
IDH
Isocitrate dehydrogenase
IM
Inner membrane
iTRAQ
isobaric tags for relative and absolute quantitation
KAT
3-Ketoacyl-CoA thiolase
KDSB
3-deoxy-manno-octulosonate cytidylyltransferase synthetase
KDO
3-Deoxy-D-manno-octulosonic Acid
MIRO
Mitochondrial RhO type GTPase
Mt
Mitochondria
NSAF
Normalised spectral abundance factor
OM
Outer membrane
OM64
Outer membrane 64kDa
PECT
Phosphorylethanolamine-phosphate cytidylyl transferase
RbcL
Ribulose-1,5-bisphosphate carboxylase oxygenase large subunit
RISP
Rieke iron-sulfur protein
RFP
Red fluorescent protein
TOM
Translocase of outer membrane
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VDAC
Voltage dependent anion channel
Q-TOF
Quadrapole time of flight
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References
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Figure legends
Figure 1. Experimental strategy used to determine the outer mitochondrial membrane
proteome. Three samples were analyzed to determine a putative outer membrane proteome
which was subsequently evaluated by a combination of prior knowledge, green fluorescent
protein localization and In vitro protein uptake experiments. (A) Each sample was derived
from a crude organelle preparation prepared from Arabidopsis cell culture. Desired fraction
(Red – Mitochondrial outer membrane) Isolated mitochondria in hypo-osmotic solution were
subjected to mechanical disruption. The inner and outer membranes were separated by
sucrose gradient centrifugation. Two samples were retained from this procedure - enriched
mitochondrial outer membranes and enriched mitochondrial inner membranes (Intra
Mitochondrial Contaminants - Blue). Extra Mitochondrial Contaminants (Green – High speed
pellet outer membrane) were enriched by subjecting the untreated crude organelle isolation
to the same sucrose gradient enrichment process used to enrich mitochondrial outer
membrane. (B) Each of these samples was analyzed by mass spectrometry and the
abundance of constituent proteins compared. (C) Statistical analysis of this data yielded a
putative outer membrane proteome. This putative proteome was further refined by the use of
a second quantitative technique – iTRAQ which included an additional sample designed to
eliminate matrix contaminants from the putative outer membrane. Additional evidence from
previous literature was used to confirm or reject members of this refined list. Identifications
for which no rigorous prior evidence existed were independently confirmed or rejected by the
used of green fluorescent protein localization and / or In vitro protein import experiments.
Figure 2. Assessment of enrichment of mitochondria from Arabidopsis suspension
cell cultures. (A) 1D SDS-PAGE separation of three fractions isolated at different stages of
mitochondrial enrichment. HSP - High Speed Pellet is the result of lysing Arabidopsis
thaliana suspension cell cultures and pelleting the insoluble fraction. Percoll Grad mitochondrial enrichment from the high speed pellet by application of the insoluble fraction to
two serial Percoll density gradient centrifugations, preserving the fractions most enriched in
mitochondria. In order to obtain our most enriched sample, the gradient enriched
mitochondrial fraction was again fractionated by surface charge utilizing Free Flow
Electrophoresis (FFE) with the fractions most enriched in mitochondria being retained. (B)
Assessment of enrichment of mitochondrial fractions by western blot. TOM40-1
(At3g20000.1) is a mitochondrial outer membrane marker, KAT2 (At2g33150.1) is a
peroxisomal marker, CALNEXIN (At5g61790.1, At5g07340.1) is present in the endoplasmic
33 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
reticulum and the large subunit of ribulose bisphosphate carboxylase (AtCg00490.1) is a
soluble plastid protein. (C) Assesment of plastid contamination of mitochondrial fractions by
western blot. Ps plastids - fraction enriched in plastids isolated from Pisum sativum, At
plastids - fraction enriched in plastids from Arabidopsis. Light harvesting complex B subunit
(LhcB) - located on the plastid thylakoid membrane.
Figure 3. Assessment of enrichment of mitochondrial outer membrane and
contaminants. (A) 1D SDS-PAGE separation of contaminant and purified outer membrane
fractions. HSP-OM - high speed pellet outer membrane. Mitoplast - mitochondria reduced in
outer membrane. Mt IM - mitochondrial inner membrane. Mt OM - mitochondrial outer
membrane Indicated band identifications were conducted by ESI-MS/MS and details can be
found in Supplementary Table VII (B) Western blot assessment of the experimental fractions
used for semi quantitative analysis by mass spectrometry. TOM40-1 (At3g20000.1) is a
mitochondrial outer membrane marker, pyruvate dehydrogenase subunit E1α (PDH E1α) is
present in the mitochondrial matrix, COXII (AtMg00160.1) is on the matrix side of the inner
mitochondrial membrane, CALNEXIN (At1g56340) is present on the endoplasmic reticulum
membrane and KAT2 (At2g33150) is a soluble peroxisomal marker. These membranes were
also probed with additional antibodies - Translocase of outer chloroplast envelope 159 kDa
subunit TOC159, TOC75, Translocase of inner chloroplast membrane 40 kDa subunit
(TIC40) and Peroxisomal membrane protein 22 but each of these antibodies failed to detect
appropriately sized products.
Figure 4. GFP tagging of mitochondrial outer membrane proteins to confirm intraorganelle localization. Arabidopsis thaliana suspension cell culture transiently transformed
with mitochondrial red fluorescent protein control and green fluorescent protein tagged
experimental proteins (A) FRO1:GFP - inner mitochondrial membrane protein (B) IDH6:GFP
- mitochondrial matrix located protein (C) ELM1:GFP - outer mitochondrial membrane
protein
(D) Example of novel mitochondrial outer membrane protein displaying similar
fluorescence pattern to ELM1, identified by mass spectrometry and location confirmed by
fluorescence microscopy. (E) GFP only.
Figure 5. Immunodetection of isolated mitochondria treated with proteinase K. (A)
TOM20-3 is a is a mitochondrial outer membrane anchored protein with a large cytosolic
domain and as such is vulnerable to digestion by external application of proteinase K. (B)
KDSB and (C) PECT1 are novel outer membrane proteins identified in the course of this
study by mass spectrometry and confirmed by green fluorescent protein localization. The
breakdown of KDSB is similar to that of TOM20-3 indicating that it is also exposed to the
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
cytoplasm. PECT1 is broken down by external application of proteinase K but appears to be
more resistant than TOM20 and KDSB. (D) SAM50 and (E) TOM40 are β -barrel outer
membrane proteins and as such are embedded in the membrane rendering them resistant to
digestion. (F) RISP is found in the cytochrome bc1 complex in the mitochondrial inner
membrane. It preservation indicates that the integrity of the inner membrane was maintained
throughout the proteinase dilution series.
Figure 6. Transient expression of several fluorescently tagged fusion proteins causes
apparent alterations in mitochondrial morphology / distribution. (A) 3-deoxy-mannooctulosonate cytidylyltransferase (KDSB) - Cells transformed with this GFP tagged protein
consistently displayed fewer but larger mitochondria than was the observed norm for
mitochondrial outer membrane proteins. (B) phosphorylethanolamine cytidylyltransferase
(PECT-1) - mitochondria bunch tightly together at one pole of the cell. (C) NADH:cytochrome
B5 reductase - mitochondrial population has an abnormal size distribution with many large
mitochondria. (D) Unknown protein - commonly observed mitochondrial morphology for
comparison.
Figure 7. Co-expression of genes encoding outer mitochondrial membrane proteins.
Co-expression of genes encoding outer mitochondrial membrane proteins. Publically
available microarrays analyzing wild-type tissues in the AtGenExpress developmental set
(Schmid et al. 2005, E-AFMX-9) and during germination shows highest expression of genes
encoding inner membrane (IM) and outer membrane (OM) proteins during germination. A)
The expression of genes encoding both inner and outer membrane proteins were analyzed
during germination (Har – seeds harvested on day of collection from WT plants; 0h – seeds
15 days after ripening, prior to imbibition; S – hours in cold stratification in the dark; SL –
hours into continuous light after 48 h stratification). Expression levels were made relative to
maximum expression, hierarchically clustered (Euclidean distance and average linkage
clustering) and 5 clusters were observed (C1-5). A group of genes showing transient
expression are indicated in the yellow box. B) Expression of genes encoding outer
mitochondrial membrane proteins only during germination. The percentage of genes in each
cluster was compared for genes encoding inner/outer membrane proteins. The significant
enrichment (p<0.001) of genes encoding OM proteins in Cluster 4 is indicated by *. Details
for each of the tissues analyzed by microarrays are shown in Supplementary Table VI.
35 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Tables
Table 1. Proteins confirmed to be in the mitochondrial outer membrane. Columns from
left to right are: AGI # (www.Arabidopsis.org), Description (Common identifier), Raw Counts
This column is a tally of the total number of identifying ions summed from three biological
replicates from the outer membrane sample followed by the High speed pellet outer
membrane then the Inner membrane sample, Ratio OM:HSP-OM This column consists of
the number of identifying ions identified in the outer membrane sample divided by the
number found in the high speed pellet outer membrane sample, p-value contains the pvalue applying to the magnitude of change (Supplementary table 3 & 4) as calculated using
Poisson regression analysis, Ratio OM:IM This column consists of the number of identifying
ions identified in the outer membrane sample divided by the number found in the inner
membrane sample, P value contains the p value applying to the magnitude of change
(Supplementary table 3 & 4) as calculated using Poisson regression analysis, iTRAQ These
three columns contain the ratios of reporter ions in IM/OM Inner membrane divided by outer
membrane Whole/OM Whole mitoplasts (mitochondria with outer membrane ruptured but
inner membrane intact) divided by outer membrane HSP-OM/OM High speed pellet outer
membrane divided by outer membrane values reported here are significant according criteria
detailed in the results section, GFP Contains the interpretation of sub cellular localization
resulting from fluorescence microscopy PMtOM refers to a sole mitochondrial outer
membrane localization whilst PMtOM++ refers to a localization on the mitochondrial outer
membrane and other locations, Reference Contains references to previously published
location information. A single protein, At5g05520.1 (SAM50-2) demonstrated enrichment in
the mitchondrial outer membrane fractions but didn't display specific targeting when tagged
with GFP but was included in this table due to evidence gathered from protease treated
mitochondria (Figure 5)
Table 2. Proteins found not to be in the mitochondrial outer membrane. From the initial
list of 64 proteins found to be enriched in the outer membrane sample, 22 were excluded
from the outer membrane proteome by one or more lines of additional evidence. Columns
from left to right are: Location indicates the subcellular compartmentalization interpretation
AGI # (www.Arabidopsis.org), Description Common identifier, Raw Counts This column is
a tally of the total number of identifying ions summed from three biological replicates from
the outer membrane sample followed by the high speed pellet outer membrane then the
Inner membrane sample, Ratio OM:HSP-OM This column consists of the number of
identifying ions identified in the outer membrane sample divided by the number found in the
high speed pellet outer membrane sample, p-value contains the p-value applying to the
magnitude of change (Supplementary table I & II) as calculated using Poisson regression
36 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
analysis, Ratio OM:IM This column consists of the number of identifying ions identified in the
outer membrane sample divided by the number found in the inner membrane sample, P
value contains the p value applying to the magnitude of change (Supplementary table I & II)
as calculated using Poisson regression analysis, iTRAQ These three columns contain the
ratios of reporter ions in IM/OM Inner membrane divided by outer membrane Whole/OM
Whole mitoplasts (mitochondria with outer membrane ruptured but inner membrane intact)
divided by outer membrane HSP-OM/OM High speed pellet outer membrane divided by
outer membrane values reported here are significant according criteria detailed in the results
section, GFP Location Contains the interpretation of sub cellular localization resulting from
fluorescence microscopy In vitro import result indicates the mitochondrial localization of
tested proteins as determined by reliance on presence of inner membrane electrochemical
gradient. Previous published location Indicates the location of previously studied proteins
Literature Contains references to previously published location information. The final protein
in Table 2, At4g17140.2 was found to be abundant and strongly enriched in the
mitochondrial outer membrane samples however confirmation of localization independent of
the organelle fractionation was attempted but not obtained. 37 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Supplementary figures
Supplementary figure 1. Members of the mitochondrial outer membrane proteome as
shown by fluorescent microscopy of transiently transformed Arabidopsis cell culture or onion
cells with full length coding sequences fused to green fluorescent protein. A mitochondrial
localization control consisting of red fluorescent protein fused to the targeting sequence of
the soybean alternative oxidase was co transformed with the experimental GFP construct.
Gene Description –TAIR (http://www.arabidopsis.org) description, Location interpretation –
sub mitochondrial localization description derived from the fluorescence microscopy results.
Transformed material – either Arabidopsis suspension cell culture or onion epidermal cell
mono layers. Three images are included in each panel; a mitochondrial RFP image showing
location of mitochondria in the cell, an experimental GFP showing the localization of the
experimental fusion protein and a composite image overlaying the RFP and the GFP data. A
hydropathy
plot
generated
by
the
DAS
trans
membrane
prediction
server
(http://www.sbc.su.se/~miklos/ ) is included. Some panels also contain in vitro protein import
assays that consist of five SDS-PAGE lanes. The first of these contains only the
radiolabelled precursor protein; the second contains radiolabelled precursor protein which
has been incubated with isolated mitochondria under conditions permissive of protein import
for 20 minutes. The third lane is the same as the second with the exception that after the
completion of the import it has been treated with proteinase k to digest any precursor
external to the mitochondria, The fourth lane is a duplicate of the second with the exception
that the import reaction also contains valinomycin which dissipates the membrane potential
across the inner mitochondrial membrane required for translocation of precursors into the
matrix. The fifth lane is a duplicate of the fourth with a proteinase K treatment following
import.
Supplementary figure 2. Members of the mitochondrial outer membrane proteome which
are also found in other subcellular locations as shown by fluorescent microscopy of
transiently transformed Arabidopsis cell culture or onion cells with full length coding
sequences fused to green fluorescent protein. A mitochondrial localization control consisting
of red fluorescent protein fused to the targeting sequence of the soybean alternative oxidase
was co transformed with the experimental GFP construct. Gene Description – TAIR
(http://www.arabidopsis.org)
description,
Location
interpretation
–
sub-mitochondrial
localization description derived from the fluorescence microscopy results. Transformed
material – either Arabidopsis suspension cell culture or onion epidermal cell mono layers.
Three images are included in each panel; a mitochondrial RFP image showing location of
mitochondria in the cell, an experimental GFP showing the localization of the experimental
38 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
fusion protein and a composite image overlaying the RFP and the GFP data. A hydropathy
plot generated by the DAS trans membrane prediction server (http://www.sbc.su.se/~miklos/ ) is included.
Supplementary figure 3. Members of the putative mitochondrial outer membrane proteome
which have been shown to be mitochondrial but not outer membrane by fluorescent
microscopy of transiently transformed Arabidopsis cell culture or onion cells with full length
coding sequences fused to green fluorescent protein. A mitochondrial localization control
consisting of red fluorescent protein fused to the targeting sequence of the soybean
alternative oxidase was co transformed with the experimental GFP construct. Gene
Description – TAIR (http://www.arabidopsis.org) description, Location interpretation –
sub-mitochondrial localization description derived from the fluorescence microscopy results.
Transformed material – either Arabidopsis suspension cell culture or onion epidermal cell
mono layers. Three images are included in each panel; a mitochondrial RFP image showing
location of mitochondria in the cell, an experimental GFP showing the localization of the
experimental fusion protein and a composite image overlaying the RFP and the GFP data. A
hydropathy
plot
generated
by
the
DAS
trans
membrane
prediction
server
(http://www.sbc.su.se/~miklos/ ) is included. Some panels also contain In vitro protein import
assays which consist of five SDS-PAGE lanes. The first of these contains only the
radiolabelled precursor protein, the second contains radiolabelled precursor protein which
has been incubated with isolated mitochondria under conditions permissive of protein import
for 20 minutes. The third lane is the same as the second with the exception that after the
completion of the import it has been treated with proteinase k to digest any precursor
external to the mitochondria, The fourth lane is a duplicate of the second with the exception
that the import reaction also contains valinomycin which dissipates the membrane potential
across the inner mitochondrial membrane required for translocation of precursors into the
matrix. The fifth lane is a duplicate of the fourth with a proteinase K treatment following
import.
Supplementary figure 4. Members of the putative mitochondrial outer membrane proteome
which have been shown not to be mitochondrial by fluorescent microscopy of transiently
transformed Arabidopsis cell culture or onion cells with full length coding sequences fused to
green fluorescent protein. A mitochondrial localization control consisting of red fluorescent
protein fused to the targeting sequence of the soybean alternative oxidase was co
transformed
with
the
experimental
(http://www.arabidopsis.org)
description,
GFP
construct.
Location
Gene
interpretation
Description
–
–
TAIR
sub-mitochondrial
localization description derived from the fluorescence microscopy results. Transformed
39 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
material – either Arabidopsis suspension cell culture or onion epidermal cell mono layers.
Three images are included in each panel; a mitochondrial RFP image showing location of
mitochondria in the cell, an experimental GFP showing the localization of the experimental
fusion protein and a composite image overlaying the RFP and the GFP data. A hydropathy
plot generated by the DAS trans membrane prediction server (http://www.sbc.su.se/~miklos/ ) is included. Some panels also contain in vitro protein import assays which consist of five
SDS-PAGE lanes. The first of these contains only the radiolabelled precursor protein, the
second contains radiolabelled precursor protein which has been incubated with isolated
mitochondria under conditions permissive of protein import for 20 minutes. The third lane is
the same as the second with the exception that after the completion of the import it has been
treated with proteinase k to digest any precursor external to the mitochondria, The fourth
lane is a duplicate of the second with the exception that the import reaction also contains
valinomycin which dissipates the membrane potential across the inner mitochondrial
membrane required for translocation of precursors into the matrix. The fifth lane is a
duplicate of the fourth with a proteinase K treatment following import.
Supplementary Figure 5. Co-expression of genes encoding outer mitochondrial membrane
proteins. Publically available microarrays analysing wild-type tissues in the AtGenExpress
developmental set (Schmid et al. 2005, E-AFMX-9) and during germination for genes
encoding inner membrane (IM) and outer membrane (OM) proteins. Germination included;
Har – seeds harvested on day of collection from WT plants; 0h – seeds 15 days after
ripening, prior to imbibition; S – hours in cold stratification in the dark; SL – hours into
continuous light after 48 h stratification. Expression levels were made relative to maximum
expression and hierarchically clustered (Euclidean distance and average linkage clustering).
Details for each of the tissues analyzed by microarrays are shown in Supplementary Table V
and the list of genes annotated as encoding inner membrane proteins are in Supplementary
Table VI.
40 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Supplementary Tables
Supplementary Table I. List of the 185 proteins detected in two or more of the outer
membrane enriched biological replicates AGI # (www.Arabidopsis.org). OM1-3
summary of the number of spectra observed which match to members of the putative outer
membrane proteome in three biological replicates of the outer membrane enriched samples
HSP-OM1-3 summary of the number of spectra observed which match to members of the
putative outer membrane proteome in three biological replicates of the prefractionation
sample designed to control for extra mitochondrial contaminants IM1-3 summary of the
number of spectra observed which match to members of the putative outer membrane
proteome in three biological replicates of the prefractionation sample designed to control for
intra mitochondrial contaminants OM:HSP-OM Change This column describes the
magnitude and direction of change between the number of spectral counts for each member
of the putative outer membrane proteome in the outer membrane enriched samples
compared to the HSP:OM samples as reported by Poisson regression analysis using the
statistical package R . It should be noted that in cases where zero counts were present, the
parameters of the statistical model were violated and the change was not reported as
significant. OM:HSP-OM p-val The p- value associated with this change OM:IM Change This
column describes the magnitude and direction of change between the number of spectral
counts for each member of the putative outer membrane proteome in the outer membrane
enriched samples compared to the inner membrane samples % coverage percentage of the
protein for which identifying mass spectra were detected. This figure is calculated by
collating all of the unlabeled LC/MS runs from the entire experiment and searching the
Arabidopsis TAIR 9 database as described in the methods section Number unique This
column contains the number of unique identifying mass spectra gathered for each member
of the putative outer membrane proteome from the same data set as the percentage
coverage.
Supplementary Table II List of the 64 members of the putative outer membrane proteome
with evidence for identification and enrichment in the outer membrane fraction AGI #
(www.Arabidopsis.org). OM1-3 summary of the number of spectra observed which match to
members of the putative outer membrane proteome in three biological replicates of the outer
membrane enriched samples HSP-OM1-3 summary of the number of spectra observed
which match to members of the putative outer membrane proteome in three biological
replicates of the pre fractionation sample designed to control for extra mitochondrial
contaminants IM1-3 summary of the number of spectra observed which match to members
41 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
of the putative outer membrane proteome in three biological replicates of the prefractionation
sample designed to control for intra mitochondrial contaminants OM:HSP-OM Change This
column describes the magnitude and direction of change between the number of spectral
counts for each member of the putative outer membrane proteome in the outer membrane
enriched samples compared to the HSP:OM samples as reported by Poisson regression
analysis using the statistical package R . It should be noted that in cases where zero counts
were present, the parameters of the statistical model were violated and the change was not
reported as significant. OM:HSP-OM p-val The p-value associated with this change OM:IM
Change This column describes the magnitude and direction of change between the number
of spectral counts for each member of the putative outer membrane proteome in the outer
membrane enriched samples compared to the inner membrane samples % coverage
percentage of the protein for which identifying mass spectra were detected. This figure is
calculated by collating all of the unlabeled LC/MS runs from the entire experiment and
searching the Arabidopsis TAIR 9 database as described in the methods section Number
unique This column contains the number of unique identifying mass spectra gathered for
each member of the putative outer membrane proteome from the same data set as the
percentage coverage.
Supplementary Table III. Summary of the location evidence gathered in the course of
this research. Columns from left to right Description (Common identifier), Spectral counting
Putative OM proteome The complete list of 64 proteins identified as enriched in the outer
membrane fractions, iTRAQ experiment proteins excluded by evidence gathered using this
technique, Literature survey a summary of positive and negative evidence relating to
members of the putative outer membrane proteome, GFP List of putative outer membrane
proteins which have been fused with GFP and used in sub cellular localization studies, In
vitro List of proteins for which sub mitochondrial localization data has been gathered in the
form of in vitro imports, Reference List of publications containing subcellular localization
evidence for members of the putative outer membrane proteome, Evidence type a
description of the nature of the experiments in the referenced publications.
Supplementary Table IV Summary of data gathered from iTRAQ labeled analysis of the
outer membrane enriched and three pre fractionation samples. Only data relevant to the
those The 114 reporter ion corresponds to the outer membrane enriched sample, 115 to the
Inner membrane sample, 116 to the whole mitoplast sample and 117 to the HSP-OM sample
AGI # (www.Arabidopsis.org), Weighted ratio of reporter ion measured in the outer
membrane enriched sample to the pre-fractionation sample (lower numbers mean more
42 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
enriched in outer membrane) Geo St Dev The geometric standard deviation calculated as
per the methods section, NN stands for Non Normal distribution and this data was discarded
N the number of unique identifying peptides used to calculate these figures.
Supplementary Table V. Details of the germination microarrays and developmental tissue
set (Schmid et al., 2005; E-AFMX-9). The ATGE accessions, number of biological
replications, genotype, sample details, age, photoperiod and growth substrate are shown.
The array name/number (no.) and tissue/developmental stage as it appears on Figure 7 is
also shown.
Supplementary Table VI. Details of the genes defined as encoding inner mitochondrial
membrane proteins. The AGI, Gene Model Description and type (source:TAIR) is shown for
each gene.
Supplementary table VII. Details of polypeptide identifications resulting from the in gel
digestion of bands excised from the gel pictured in figure 3A
Supplementary methods. Detailed mass spectrometry methods.
43 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Table 1. Proteins confirmed to be in the mitochondrial outer membrane. Columns from left to right are: AGI # (www.Arabidopsis.org), Description (Common identifier), Raw Counts This column is a tally of the total
number of identifying ions summed from three biological replicates from the outer membrane sample followed by the High speed pellet outer membrane then the Inner membrane sample, Ratio OM:HSP-OM This column
consists of the number of identifying ions identified in the outer membrane sample divided by the number found in the high speed pellet outer membrane sample, p-value contains the p-value applying to the magnitude of
change (Supplementary table 3 & 4) as calculated using Poisson regression analysis, Ratio OM:IM This column consists of the number of identifying ions identified in the outer membrane sample divided by the number
found in the inner membrane sample, P value contains the p value applying to the magnitude of change (Supplementary table 3 & 4) as calculated using Poisson regression analysis, iTRAQ These three columns contain
the ratios of reporter ions in IM/OM Inner membrane divided by outer membrane Whole/OM Whole mitoplasts (mitochondria with outer membrane ruptured but inner membrane intact) divided by outer membrane HSPOM/OM High speed pellet outer membrane divided by outer membrane values reported here are significant according criteria detailed in the results section, GFP Contains the interpretation of sub cellular localization
resulting from fluorescence microscopy PMtOM refers to a sole mitochondrial outer membrane localization whilst PMtOM++ refers to a localization on the mitochondrial outer membrane and other locations, Reference
Contains references to previously published location information. A single protein, At5g05520.1 (SAM50-2) demonstrated enrichment in the mitchondrial outer membrane fractions but didn't display specific targeting when
tagged with GFP but was included in this table due to evidence gathered from protease treated mitochondria (Figure 5)
AGI #
Raw Counts
Description
Spectral counting
TOM9-­‐2
TraB family protein
myosin related
unknown protein
TOM20-­‐2
TOM6
HXKL-­‐1
KDSB
ATP binding / kinase
Metaxin
HXK2
FAC1
PECT1
VDAC1
SAM50-­‐1
TOM40-­‐1
HXK4
TOM20-­‐3
unknown protein
BCS-­‐1
BCS-­‐1 like
unknown protein
MIRO2
HXK1
SAM50-­‐2
TOM5
OM64
DGS1
VDAC3
NADH:cytochrome B5 Reductase
ELM1
MIRO1
TOM20-­‐4
TOM9-­‐1
unknown protein
VDAC4
Anion-­‐transport
VDAC2
OM:HSP-­‐OM:IM
3:0:0
41:7:0
53:13:0
13:3:0
6:0:0
11:2:0
41:22:0
53:24:16
8:0:0
14:6:1
92:71:25
7:2:0
55:28:0
90:50:53
53:24:3
93:42:12
13:4:0
23:0:0
44:13:0
29:13:0
10:4:0
30:5:0
7:0:0
124:97:31
32:8:13
21:12:8
34:13:0
72:23:3
97:63:63
34:7:0
17:6:0
89:55:8
8:0:0
15:5:4
50:19:0
80:36:37
13:5:0
122:77:64
Ratio OM:HSP-­‐OM
AT1G04070.1
AT1G05270.1
AT1G06530.1
AT1G24267.1
AT1G27390.1
AT1G49410.1
AT1G50460.1
AT1G53000.1
AT2G01460.1
AT2G19080.1
AT2G19860.1
AT2G38280.1
AT2G38670.1
AT3G01280.1
AT3G11070.1
AT3G20000.1
AT3G20040.1
AT3G27080.1
AT3G27930.1
AT3G50930.1
AT3G50940.1
AT3G58840.1
AT3G63150.1
AT4G29130.1
AT5G05520.1
AT5G08040.1
AT5G09420.1
AT5G12290.1
AT5G15090.1
AT5G17770.1
AT5G22350.1
AT5G27540.1
AT5G40930.1
AT5G43970.1
AT5G55610.1
AT5G57490.1
AT5G60730.1
AT5G67500.1
AT5G16870.1
AT5G20520.1
AT4G35970.1
AT5G39410.1
aminoacyl-­‐tRNA hydrolase
WAV2
APX5
unknown protein
16:6:0
16:3:0
10:4:0
52:34:3
2.7
5.3
2.5
1.5
∞
5.9
4
4.3
∞
5.5
1.9
2.2
∞
2.3
1.3
3.5
2
1.8
2.2
2.2
3.2
∞
3.4
2.2
2.5
6
∞
1.3
4
1.75
2.6
3.2
1.5
4.9
2.8
1.6
∞
3
2.6
2.2
2.6
1.6
Ratio OM:IM
P value
^
∞
1.50E-­‐05
∞
5.60E-­‐06
∞
0.022
∞
^
∞
0.027
∞
0.018
∞
0.001
3.3
^
∞
0.082
14
0.101
3.7
0.118
∞
0.004
∞
0.001
1.7
0.001
17.7
1.90E-­‐05
7.7
0.039
∞
^
∞
0.0001
∞
0.016
∞
0.121
∞
0.0002
∞
^
∞
0.07
4
0.0004
2.5
0.122
2.6
0.003
∞
0.0000019
24
0.008
1.5
0.0001
∞
0.028
∞
0.005
11.1
^
∞
0.033
3.75
0.0003
∞
0.00007
2.2
0.07
∞
0.002
1.9
Outer membrane and other
0.04
∞
0.008
∞
0.121
∞
0.054
17.3
P value
^
^
^
^
^
^
^
0.00003
^
0.011
8.00E-­‐09
^
^
0.002
1.30E-­‐06
2.4E-­‐11
^
^
^
^
^
^
^
1.2E-­‐12
0.006
0.02
^
7E-­‐08
0.008
^
^
7E-­‐11
^
0.019
^
0.0001
^
0.00003
^
^
^
0.0000015
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
IM/OM
iTRAQ
Whole/O
M
GFP Location
Reference
CON/OM
Werhahn et al., 2001
0.285
0.462
PMtOM
P MtOM
PMtOM
Werhahn et al., 2001
Werhahn et al., 2001
0.305
0.165
0.229
0.276
0.255
0.264
0.233
0.27
0.564
0.217
0.254
0.511
0.274
0.417
0.31
0.429
0.395
PMtOM
PMtOM
P MtOM
Lister et al., 2007
P MtOM
P MtOM
P MtOM
Lee et al., 2009
P MtOM
Werhahn et al., 2001
P MtOM
Werhahn et al., 2001
0.162
0.231
0.213
0.23
0.203
0.211
0.201
0.422
0.185
0.377
0.195
0.446
0.234
0.372
0.2
0.214
P MtOM
PMtOM
PMtOM
PMtOM
P MtOM
P MtOM
See legend
Werhahn et al., 2001
Chew et al., 2004
Xu et al., 2008
Lee et al., 2009
P MtOM
P MtOM
Arimura et al., 2008
Werhahn et al., 2001
Werhahn et al., 2001
P MtOM
0.187
0.249
0.199
Lee et al., 2009
P MtOM
Lee et al., 2009
0.464
PMtOM++
PMtOM++
PMtOM++
PMtOM++
Table 2. Proteins found not to be in the mitochondrial outer membrane. From the initial list of 64 proteins found to be enriched in the outer membrane sample, 22 were excluded from the outer membrane proteome by one or more lines of additional evidence. Columns from left to right are: Location
indicates the subcellular compartmentalization interpretation AGI # (www.Arabidopsis.org), Description Common identifier, Raw Counts This column is a tally of the total number of identifying ions summed from three biological replicates from the outer membrane sample followed by the High speed pellet outer
membrane then the Inner membrane sample, Ratio OM:HSP-OM This column consists of the number of identifying ions identified in the outer membrane sample divided by the number found in the high speed pellet outer membrane sample, p-value contains the p-value applying to the magnitude of change
(Supplementary table 3 & 4) as calculated using Poisson regression analysis, Ratio OM:IM This column consists of the number of identifying ions identified in the outer membrane sample divided by the number found in the inner membrane sample, P value contains the p value applying to the magnitude of
change (Supplementary table II & III) as calculated using Poisson regression analysis, iTRAQ These three columns contain the ratios of reporter ions in IM/OM Inner membrane divided by outer membrane Whole/OM Whole mitoplasts (mitochondria with outer membrane ruptured but inner membrane intact)
divided by outer membrane HSP-OM/OM High speed pellet outer membrane divided by outer membrane values reported here are significant according criteria detailed in the results section, GFP Location Contains the interpretation of sub cellular localization resulting from fluorescence microscopy in vitro
import result indicates the mitochondrial localization of tested proteins as determined by reliance on presence of inner membrane electrochemical gradient. Previous published location Indicates the location of previously studied proteins Reference Contains references to previously published location
information. The final protein in Table 2, At4g17140.2 was found to be abundant and strongly enrighed in the mitochondrial outer membrane samples however confirmation of localization independent of the organelle fractionation was attempted but not obtained.
Location
Mitochondrial
Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
No further evidence
AGI #
Description
Raw counts
AT2G30970.1
AT5G20080.1
AT1G75200.1
AT2G26240.1
AT2G45870.1
AT3G63520.1
AT4G00355.1
AT4G27760.1
AT4G33360.1
AT5G67590.1
AT3G09810.1
AT3G22200.1
AT5G15640.1
AT4G04180.1
AT1G61570.1
AT1G68680.1
AT3G46560.1
AT3G52730.1
AT4G16450.1
AtMg01080.1
AT4G35260.1
AT4G17140.2
ASP1
NADH-­‐cytochrome b5 reductase, putative
flavodoxin family protein
unknown protein
Bestrophin-­‐like protein
CCD1
unknown protein
FEY
terpene cyclase/mutase-­‐related
FRO1
IDH6
GABA aminotransferase
mitochondrial substrate carrier family protein
unknown protein
TIM13
unknown protein
TIM9
UQCRX-­‐like family protein
unknown protein
subunit 9 of mitochondrial F0-­‐ATPase IDH1
unknown protein
OM:HSP-­‐OM:IM
23:0:9
30:9:4
22:7:0
9:0:0
4:0:0
40:20:2
8:0:0
4:0:0
10:0:0
5:0:0
17:0:4
47:13:29
7:0:0
10:0:0
14:3:0
8:0:0
4:0:0
8:0:0
5:0:0
4:0:0
24:0:6
220:104:21
Spectral counting
Ratio Ratio OM:HSP-­‐
OM:IM
P value
OM
^
2.6
∞
3.3
0.002
7.5
3.1
0.008
∞
^
∞
∞
^
∞
∞
2
0.011
20
^
∞
∞
^
∞
∞
^
∞
∞
^
∞
∞
^
4.2
∞
3.6
0.00004
1.6
^
∞
∞
^
∞
∞
4.7
0.015
∞
^
∞
∞
^
∞
∞
^
∞
∞
^
∞
∞
^
∞
∞
^
4
∞
2.1
1.5E-­‐07
10.5
iTRAQ
P value
0.017
0.00015
^
^
^
0.000035
^
^
^
^
0.009
0.041
^
^
^
^
^
^
^
^
0.002
6.22E-­‐27
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IM/OM
1.229
0.713
Whole/OM
1.284
0.486
0.128
0.51
0.802
0.776
0.203
In vitro import result
Previous published location
Reference
CON/OM
0.611
0.341
0.557
GFP Location
0.403
0.32
Inner membrane or matrix
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Not Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
See table legend
Inner Membrane -­‐ Complex I
Meyer et al. 2008
Inter Membrane Space
Inner Membrane -­‐ Complex I
Inter Membrane Space
Inner Membrane -­‐ Complex III
Inner Membrane -­‐ Complex I
Inner Membrane -­‐ Complex V
Mitochondrial Matrix
Lister et al. 2004
Meyer et al. 2008
Lister et al. 2004
Meyer et al. 2008
Klodmann et al. 2010
Meyer et al. 2008
Zhao, 1996
Inner membrane or matrix
Inner membrane or matrix
Inner membrane or matrix
A
B
C
Mass
Spectrometry
Purification of Fractions
Verification
Disruption
Soluble
Mitochondrial enrichment
Membrane Separation
Dense charge
Dense
Statistical Cut Offs
Dense
Less dense charge
Membrane
Less dense
Less dense
Soluble
Organelles
Outer
Membrane
Dense
(
Intra Mitochondrial
Contaminants
(
Mitochondrial
Outer Membrane
(
Extra Mitochondrial
Contaminants
t
en l
nd nta
pe e es
de rim u
In xpe niq
e ech
T
Mitochondria
Inner
Membrane
e
r
io dg
Pr wle
o
Kn
Organelles
Mitochondria
Insoluble
Putative Proteome
Insoluble
Membrane
Less dense
Figure 1. Experimental strategy used to determine the outer mitochondrial membrane proteome. Three samples were
analyzed to determine a putative outer membrane proteome which was subsequently evaluated by a combination of prior
knowledge, green fluorescent protein localization and In vitro protein uptake experiments. (A) Each sample was derived from
a crude organelle preparation prepared from Arabidopsis cell culture. Desired fraction (Red – Mitochondrial outer membrane)
Isolated mitochondria in hypo-osmotic solution were subjected to mechanical disruption. The inner and outer membranes were
separated by sucrose gradient centrifugation. Two samples were retained from this procedure - enriched mitochondrial outer
membranes and enriched mitochondrial inner membranes (Intra Mitochondrial Contaminants - Blue). Extra Mitochondrial
Contaminants (Green – High speed pellet outer membrane) were enriched by subjecting the untreated crude organelle isolation to the same sucrose gradient enrichment process used to enrich mitochondrial outer membrane. (B) Each of these samples was analyzed by mass spectrometry and the abundance of constituent proteins compared. (C) Statistical analysis of this
data yielded a putative outer membrane proteome. This putative proteome was further refined by the use of a second quantitative technique – iTRAQ which included an additional sample designed to eliminate matrix contaminants from the putative outer
membrane. Additional evidence from previous literature was used to confirm or reject members of this refined list. Identifications for which no rigorous prior evidence existed were independently confirmed or rejected by the used of green fluorescent
protein localization and / or In vitro protein import experiments.
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E
FF
P
HS
rad
ds
sti
ll G
KAT2
LhcB
At2g05100.1
r co
At2g33150.1
34 kDa
Pe
14 kDa
ds
sti
AtCg00490.1
Pla
RbcL
20 kDa
Pla
At5g61790.1
At5g07340.1
Ps
VDAC
CALNEXIN
At
ATP
Synthase
E
30 kDa
At3g20000.1
FF
TOM40-1
66 kDa
rad
P
HS
ll G
E
FF
rad
P
HS
ll G
rco
rco
100 kDa
45 kDa
C
Pe
B
Pe
A
25 kDa
60 kDa
52 kDa
42 kDa
Figure 2. Assessment of enrichment of mitochondria from Arabidopsis suspension cell cultures. (A) 1D SDS-PAGE
separation of three fractions isolated at different stages of mitochondrial enrichment. HSP - High Speed Pellet is the result
of lysing Arabidopsis thaliana suspension cell cultures and pelleting the insoluble fraction. Percoll Grad - mitochondrial
enrichment from the high speed pellet by application of the insoluble fraction to two serial Percoll density gradient centrifugations, preserving the fractions most enriched in mitochondria. In order to obtain our most enriched sample, the gradient
enriched mitochondrial fraction was again fractionated by surface charge utilizing Free Flow Electrophoresis (FFE) with the
fractions most enriched in mitochondria being retained. (B) Assessment of enrichment of mitochondrial fractions by western
blot. TOM40-1 (At3g20000.1) is a mitochondrial outer membrane marker, KAT2 (At2g33150.1) is a peroxisomal marker,
CALNEXIN (At5g61790.1, At5g07340.1) is present in the endoplasmic reticulum and the large subunit of ribulose bisphosphate carboxylase (AtCg00490.1) is a soluble plastid protein. (C) Assesment of plastid contamination of mitochondrial
fractions by western blot. Ps plastids - fraction enriched in plastids isolated from Pisum sativum, At plastids - fraction
enriched in plastids from Arabidopsis. Light harvesting complex B subunit (LhcB) - located on the plastid thylakoid membrane.
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A
B
OM
Mt
IM
Mt
t
las
top
Mi
OM
PHS
OM
Mt
IM
Mt
t
las
top
Mi
M
-O
P
HS
TOM40-1
100 kDa
66 kDa
45 kDa
At3g20000.1
34 kDa
PDH E1α
43 kDa
ATP synthase
β subunit
At5g08670.1
At1g01090.1
30 kDa
20 kDa
14 kDa
VDAC(s)
At5g15090.1
At5g57490.1
COXII
AtMg00160.1
29 kDa
CALNEXIN
60 kDa
KAT2
48 kDa
At5g61790.1
At5g07340.1
At2g33150.1
Figure 3. Assessment of enrichment of mitochondrial outer membrane and contaminants. (A) 1D SDS-PAGE separation of contaminant and purified outer membrane fractions. HSP-OM - high speed pellet outer membrane. Mitoplast mitochondria reduced in outer membrane. Mt IM - mitochondrial inner membrane. Mt OM - mitochondrial outer membrane
Indicated band identifications were conducted by ESI-MS/MS and details can be found in supplementary table VII (B)
Western blot assessment of the experimental fractions used for semi quantitative analysis by mass spectrometry. TOM40-1
(At3g20000.1) is a mitochondrial outer membrane marker, pyruvate dehydrogenase subunit E1α (PDH E1α) is present in
the mitochondrial matrix, COXII (AtMg00160.1) is on the matrix side of the inner mitochondrial membrane, CALNEXIN
(At1g56340) is present on the endoplasmic reticulum membrane and KAT2 (At2g33150) is a soluble peroxisomal marker.
These membranes were also probed with additional antibodies - Translocase of outer chloroplast envelope 159 kDa subunit TOC159, TOC75, Translocase of inner chloroplast membrane 40 kDa subunit (TIC40) and Peroxisomal membrane
protein 22 but each of these antibodies failed to detect appropriately sized products.
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mitochondrial RFP control
(matrix)
experimental GFP
composite image
A
FRO1:GFP
mitochondrial inner
membrane control
B
IDH6:GFP
mitochondrial matrix
control
C
ELM1:GFP
mitochondrial outer
membrane control
D
At5G55610.1:GFP
example of a novel
mitochondrial outer
membrane localization
E
GFP only control
Figure 4. Example fluorescence microscopy imagery. Arabidopsis thaliana suspension cell culture transiently transformed with
mitochondrial red fluorescent protein control and green fluorescent protein tagged experimental proteins (A) FRO1:GFP - inner
mitochondrial membrane protein (B) IDH6:GFP - mitochondrial matrix located protein (C) ELM1:GFP - outer mitochondrial membrane
protein (D) Example of novel mitochondrial outer membrane protein displaying similar fluorescence pattern to ELM1, identified by mass
spectrometry and location confirmed by fluorescence microscopy. (E) GFP only
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mitochondrial RFP control
(matrix)
A
experimental GFP
composite image
KDSB At1g53000.1
CMP-KDO synthetase
B
PECT-1 At2g3867120.1
phosphorylethanolamine
cytidylyltransferase
C
ATCBR At5g17770.1
NADH:cytochrome B5
reductase
D
At5g55610.1
unknown protein
normal mitochondrial
morphology
Figure 5. High expression of several fluorescently tagged fusion proteins causes apparent alterations in mitochondrial morphology / distribution. (A) 3-deoxy-manno-octulosonate cytidylyltransferase (KDSB) - Cells transformed with this
GFP tagged protein consistently displayed fewer but larger mitochondria than was the observed norm for mitochondrial outer
membrane proteins. (B) phosphorylethanolamine cytidylyltransferase (PECT-1) - mitochondria bunch tightly together at one
pole of the cell. (C) NADH:cytochrome B5 reductase - mitochondrial population has an abnormal size distribution with many
large mitochondria. (D) Unknown protein - commonly observed mitochondrial morphology for comparison.
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N
o
P
2 k
m
g
/m
8
lP
m
k
g
/m
32
lP
k
m
g
/
m
64
lP
m
k
g
/m
lP
k
22 kDa
A
TOM20-3
At3g27080.1
B
KDSB
At1g53000.1
C
PECT1
At2g38670.1
47 kDa
D
SAM50(s)
At5g05520.1
At3g11070.1
58 kDa
57 kDa
E
TOM40-1
At3g20000.1
34 kDa
F
RISP
At5g13430.1
30 kDa
32 kDa
Figure 6. Immunodetection of isolated mitochondria treated with proteinase K. (A) TOM20-3 is a is a mitochondrial
outer membrane anchored protein with a large cytosolic domain and as such is vulnerable to digestion by external application of proteinase K. (B) KDSB and (C) PECT1 are novel outer membrane proteins identified in the course of this study by
mass spectrometry and confirmed by green fluorescent protein localization. The breakdown of these two proteins is similar
to that of TOM20-3 indicating that they are exposed to the cytoplasm. (D) SAM50 is encoded by two genes in Arabidopsis
with protein products of similar sequence but slightly differeing sizes leading to the detection of two bands with this antibody.
SAM50 and (E) TOM40 are β-barrel outer membrane proteins and as such are embedded in the membrane rendering them
resistant to digestion. (F) RISP is found in the Bc1 complex in the mitochondrial inner membrane. It preservation indicates
that the integrity of the inner membrane was maintained throughout the proteinase dilution series.
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Germination
Seedling
Flower
Leaf
Root
99
98
95
94
93
9
3
91
90
89
87
29
28
27
26
25
24
23
22
21
20
19
17
16
15
14
13
12
10
8
6
5
4
2
92
73
45
43
42
41
40
39
37
36
35
34
33
32
31
101
100
97
96
7
1
84
83
82
81
79
78
77
76
48 h SL
24 h SL
12 h SL
6 h SL
1 h SL
48 h S
12 h S
1hS
0h
Har
At3g20040.1
HXK4
At2g01460.1
ATP binding / kinase
At4g29130.1
HXK1
At3g27930.1
Porin 3 superfamily
At3g50930.1
BCS-1
At4g35970.1
APX5
At5g39410.1
unknown protein
At1g04070.1
TOM9-2
At5g20520.1
WAV2
At3g63150.1
MIRO2
At5g17770.1 NADH:cyt. B5 Reductase
At5g08040.1
TOM5
At5g60730.1
Anion-transport
At5g43970.1
TOM9-1
At3g27080.1
TOM20-3
At2g38280.1
FAC1
At1g27390.1
TOM20-2
At1g49410.1
TOM6
At3g20000.1
TOM40-1
At5g57490.1
VDAC4
At5g16870.1 aminoacyl-tRNA hydrolase
At1g05270.1
TraB family protein
At5g40930.1
TOM20-4
At5g09420.1
OM64
At5g05520.1
SAM50-2
At2g19080.1
Metaxin
At2g19860.1
HXK2
At5g27540.1
MIRO1
At2g38670.1
PECT1
At5g15090.1
VDAC3
At3g01280.1
VDAC1
At5g67500.1
VDAC2
At5g55610.1
unknown protein
At3g58840.1
unknown protein
At1g06530.1
myosin related
At5g12290.1
DGS1
At1g50460.1
HXKL-1
At1g53000.1
KDSB
Seed
0
Relative expression level
0.5
1
Figure 7. Co-expression of genes encoding outer mitochondrial membrane proteins. Publically available microarrays
analyzing wild-type tissues in the AtGenExpress developmental set (Schmid et al. 2005, E-AFMX-9) were normalized
together with microarrays analyzing expression during germination (Har – harvested on day of collection from WT plants; 0h
– 15 days after ripening, prior to imbibition; S – hours in cold stratification in the dark; SL – hours into continuous light after 48
h stratification). Expression levels were made relative to maximum expression and hierarchically clustered (Euclidean
distance and average linkage clustering). Details for each of the tissues analyzed by microarrays are shown in Supplementary
Table 6.
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