Download Multiple Lines of Evidence Localize Signaling

Multiple Lines of Evidence Localize Signaling,
Morphology, and Lipid Biosynthesis Machinery to the
Mitochondrial Outer Membrane of Arabidopsis[W][OA]
Owen Duncan, Nicolas L. Taylor, Chris Carrie, Holger Eubel, Szymon Kubiszewski-Jakubiak, Botao Zhang,
Reena Narsai A. Harvey Millar, and James Whelan*
Australian Research Council Centre of Excellence in Plant Energy Biology (O.D., N.L.T., C.C., H.E., S.K.-J.,
B.Z., R.N., A.H.M., J.W.), Centre for Comparative Analysis of Biomolecular Networks (N.L.T., A.H.M.), and
Centre for Computational Systems Biology (R.N.), University of Western Australia, Crawley, Western
Australia 6009, Australia
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 in Arabidopsis (Arabidopsis
thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated 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 that affected
mitochondrial morphology and/or segregation, a protein that 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 acid accumulation in developing seeds, were shown to be located in the
outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of
the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the
context of mitochondrial intracellular interaction.
Mitochondria are double membrane-bound organelles. While the inner membrane and its role in oxidative phosphorylation have 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). While 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
the Outer Membrane 40-kD subunit (TOM40; Jänsch
et al., 1998), and the regulators of mitochondrial distribution and morphology, mitochondrial r-type
GTPases (MIRO; Yamaoka and Leaver, 2008), the lineagespecific evolution and specialization of mitochondrial
* Corresponding author; e-mail [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 (www.plantphysiol.org) is:
James Whelan ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.183160
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 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 plastidlocalized protein, would be missed or incorrectly
annotated in an orthology-based approach comparing
mitochondrial proteins from other organisms (Chew
et al., 2004).
Using GFP tagging, mitochondria have been visualized to undergo fusion, fission, and rapid movements, suggesting a dynamic interaction with
components of the cytoskeleton (Sheahan et al., 2004,
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 with mammalian systems, different protein
components mediate this process. For example, plant
genomes do not appear to have genes encoding the
B-cell lymphoma 2 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
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Duncan et al.
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 (Camougrand
et al., 1995) in yeast and the mammalian Mitochondrial Antiviral Signaling Protein (Koshiba et al.,
2011b) are examples of such outer mitochondrial
membrane signaling components identified in other
species, none of which have thus far been localized
in plants.
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, therefore, are 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, 85% estimated coverage in S. 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 (Arabidopsis
thaliana) 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 suborganelle proteomics on low-abundance fractions.
With these challenges in mind, we developed a
statistically rigorous quantitative proteomic workflow
to provide a qualitative assessment of suborganelle
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. First,
we enriched outer membrane vesicles from highly
purified mitochondria and compared the abundance
of its constituents with that in prefractionated “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 [35S]Metlabeled precursor proteins into purified mitochondria.
By hierarchical evaluation of these 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
signaling, morphology, and defense responses.
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 subcellular localization
of proteins on the mitochondrial outer membrane.
The first aspect of this workflow was the selection of
multiple, independent prefractionated samples for
comparisons with enriched mitochondrial outer membranes in order to independently analyze the likely
causes of contaminants (Fig. 1A). Furthermore, we
sought a control for the protein composition of other
mitochondrial membranes (IM), a control for the
coenrichment of contaminating proteineous structures
in the outer membrane isolation process (high-speed
pellet-outer membrane [HSP-OM]), and a control for
nonmembrane 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 (Fig. 1B), we were
able, in effect, to subtract contaminants from the
mitochondrial outer membrane fraction in a statistically rigorous fashion (Fig. 1C). It should be noted that
the success or failure of this technique hinges not on
the purity of the fractions analyzed but on the relative
enrichment of sources of contamination in the prefractionated 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 subcellular/submitochondrial location for each of
these proteins was then assessed by techniques independent of the fractionation procedure, including GFP
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 (MS), and confirmation by another
independent approach, such as in vitro or in vivo
targeting ability to the outer membrane or westernblot analysis (Millar et al., 2009). The application of
these criteria led to the identification of a stringent set
of 42 proteins (Fig. 1C).
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Arabidopsis Mitochondrial Outer Membrane
Figure 1. Experimental strategy used to determine the outer mitochondrial membrane proteome. Three samples were analyzed
to determine a putative outer membrane proteome that was subsequently evaluated by a combination of prior knowledge, GFP
localization, and in vitro protein uptake experiments. A, Each sample was derived from a crude organelle preparation prepared
from Arabidopsis cell culture. For the desired fraction (red, mitochondrial outer membrane), isolated mitochondria in
hypoosmotic solution were subjected to mechanical disruption. The inner and outer membranes were separated by Suc gradient
centrifugation. Two samples were retained from this procedure: enriched mitochondrial outer membranes and enriched
mitochondrial inner membranes (intramitochondrial contaminants [blue]). Extramitochondrial contaminants (green [HSP-OM])
were enriched by subjecting the untreated crude organelle isolation to the same Suc gradient enrichment process used to enrich
the mitochondrial outer membrane. B, Each of these samples was analyzed by MS, and the abundance of constituent proteins
was compared. C, Statistical analysis of the 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 use of GFP localization and/or in vitro protein import experiments.
Preparation of Mitochondrial Outer Membrane and
Prefractionated 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 the HSP) and then
enriching mitochondria from this fraction, initially by
density gradient centrifugation and subsequently by
free-flow electrophoresis (FFE). The ability of FFE
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
a greater degree than single physical property isolation strategies (Fig. 2). The mitochondrial enrichment
of each of these fractions was assessed by comparison
of Coomassie blue staining profiles (Fig. 2A) and
immunodetection (Fig. 2, B and C) of proteins found
in mitochondria (TOM40), peroxisomes (3-KetoacylCoenzyme A Thiolase [KAT2]), plastids (ribulose
bisphosphate carboxylase, large subunit, and lightharvesting complex B [LhcB]), and the endoplasmic
reticulum (Calnexin; Fig. 2). Coomassie blue staining
of proteins separated by SDS-PAGE indicated that
both the gradient-fractionated and FFE-fractionated
samples were enriched in mitochondria compared
with the starting material, the HSP (Fig. 2A). Imagebased 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 higher after FFE (Fig. 2B).
Levels of endoplasmic reticulum were assessed with
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Duncan et al.
Figure 2. Assessment of the enrichment of mitochondria from Arabidopsis suspension cell cultures. A, One-dimensional SDSPAGE separation of three fractions isolated at different stages of mitochondrial enrichment. HSP is the result of lysing Arabidopsis
suspension cell cultures and pelleting the insoluble fraction. Percoll Grad represents mitochondrial enrichment from the HSP 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 FFE, with the fractions most enriched in mitochondria being retained. B, Assessment of
the 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 and At5g07340.1) is present in the endoplasmic reticulum,
and the large subunit of ribulose bisphosphate carboxylase (RbcL; AtCg00490.1) is a soluble plastid protein. C, Assessment of
plastid contamination of mitochondrial fractions by western blot. At plastids, Fraction enriched in plastids from Arabidopsis; Ps
plastids, fraction enriched in plastids isolated from pea (Pisum sativum). LhcB was located on the plastid thylakoid membrane.
an antibody raised to recombinant human Calreticulin
(which primarily detects the membrane-bound form
of this protein, Calnexin, in Arabidopsis, as evidenced
by the 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 (Fig. 2B) corresponding to the two Calnexin isoforms At5g07340.1
(61.4 kD) and At5g61790 (60.4 kD). 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 the Rubisco large subunit showed a
4-fold reduction in chloroplast contamination after
gradient purification and a 60-fold reduction following FFE (Fig. 2B). An additional membrane-bound
plastid marker, LhcB, was readily detectable in enriched plastid samples and present in the HSP fraction
but was undetectable in either of the mitochondriaenriched fractions (Fig. 2C). This high level of enrichment laid a firm foundation for the subsequent
identification of extramitochondrial contaminants
present in outer membrane fractions.
Mitochondria were then subfractionated 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 hypoosmotic 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-Elvehjem homogenizer. The protein-rich, dense inner membranes are
separated from the less dense outer membranes by
sedimentation and subsequent flotation through Suc
gradients. This membrane fractionation was performed
on two samples, the first being FFE-enriched mitochondria, to yield enriched mitochondrial outer membrane (Mt OM), and the second being the crude
organelle enrichment HSP, to yield a contaminated
outer membrane fraction (HSP-OM). Additional prefractionated 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 immunodetection of proteins characteristic of the endoplasmic reticulum, plastids, and the mitochondrial
inner membrane (Fig. 3). Coomassie Brilliant Blue
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Arabidopsis Mitochondrial Outer Membrane
staining of the separated fractions revealed differing
protein profiles for each of the fractionated samples
(Fig. 3A). Identification of several high-abundance
bands on this gel by electrospray ionization-tandem
mass spectrometry (ESI-MS/MS) showed that the
most abundant band in the mitochondrial outer membrane fraction consists of the previously identified
mitochondrial outer membrane proteins, VoltageDependent Anion Channel1 (VDAC1), -2, -3, and -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 b-subunit of ATP
synthase, which is located on the inner mitochondrial
membrane. Details of these identifications can be
found in Supplemental Table S7. Immunodetection
of the mitochondrial outer membrane marker TOM40
(At3g20000.1) in these same fractions (Fig. 3B) revealed strong enrichment of the outer membrane and
corresponding depletion in the prefractionated samples. Detection of TOM40 in the mitoplast and inner
membrane samples is a result of the incomplete
separation of outer membrane from inner membrane
during the homogenization process. Conversely, detection of the mitochondrial matrix marker Pyruvate
Dehydrogenase subunit 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 (Fig. 2B); however, KAT2, being a soluble protein, is lost in the
preparation of this membrane sample. Immunodetection 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 subfractionated samples. However the chloroplast membrane proteins TOC159 (data not shown)
and LhcB (Fig. 2C) were detected in purified chloroplasts.
Figure 3. Assessment of the enrichment of mitochondrial outer membrane and contaminants. A, One-dimensional SDS-PAGE
separation of contaminant and purified outer membrane fractions. The indicated band identifications were conducted by ESI-MS/
MS, and details can be found in Supplemental Table S7. B, Western-blot assessment of the experimental fractions used for
semiquantitative analysis by MS. TOM40-1 (At3g20000.1) is a mitochondrial outer membrane marker, pyruvate dehydrogenase
subunit E1a (PDH E1a) 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 the additional antibodies TOC159, TOC75, translocase
of inner chloroplast membrane 40-kD subunit (TIC40), and peroxisomal membrane protein 22, but each of these antibodies
failed to detect appropriately sized products.
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Duncan et al.
MS Analysis of Outer Membrane Composition
The first quantitative approach undertaken involved the counting of spectra that matched individual proteins in the purified outer membrane fraction.
These counts were then compared with those observed
in the prefractionated samples. The first of these
comparisons was designed to eliminate membraneassociated 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 (Mt OM, Mt IM, and
HSP-OM) was 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 The Arabidopsis Information Resource [TAIR] 9
data set) 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
(Supplemental Table S1).
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 two or more biological replicates and
have a P value of 0.13 or less for enrichment over both
the HSP-OM and Mt IM samples when the spectra
from all biological replicates are pooled (Supplemental
Table S2). 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 GFP-targeting studies of several proteins
falling on either side of this cutoff, 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 that they were present in more than
one biological replicate, were also included on the
basis that these were likely to be 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 nonplant species. We calculated 88% coverage of
known outer membrane mitochondrial proteins in our
preliminary data set versus 85% in S. cerevisiae (Zahedi
et al., 2006) and 67% in N. crassa (Schmitt et al., 2006).
This coverage was achieved with a pool of just 64
proteins versus 118 in S. cerevisiae. The two proteins
that were not included in this preliminary set were
TOM7 (At5g41685.1) and BIGYIN (At3g57090.1; an
Arabidopsis ortholog of Fis1 in S. cerevisiae and mammals; Scott et al., 2006).
Literature, MS, 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 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 Tables I
and II and Supplemental Table S3.
Literature
Components of the TOM complex have been isolated previously 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,
20-2, 20-3, 20-4, 9-1, 9-2, 5, and 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 I). A number
of proteins present in the putative outer mitochondrial
membrane proteome in this study have previously
been reported to be in other subcellular locations.
These proteins included TIM9 (At3g46560.1) and
TIM13 (At1g61570.1), which have been previously
reported to be soluble proteins of the mitochondrial
intermembrane space (Koehler, 2004; Lister et al.,
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Arabidopsis Mitochondrial Outer Membrane
Table I. Proteins confirmed to be in the mitochondrial outer membrane
Columns from left to right are as follows: AGI No., Arabidopsis Genome Initiative identifier (www.Arabidopsis.org); Description, common
identifier; Raw Counts, tally of the total number of identifying ions summed from three biological replicates from the outer membrane sample
followed by the HSP-OM and then the inner membrane sample; Ratio OM:HSP-OM, number of identifying ions identified in the outer membrane
sample divided by the number found in the HSP-OM sample; P, P value applying to the magnitude of change (Supplemental Tables S3 and S4) as
calculated using Poisson regression analysis; Ratio OM:IM, number of identifying ions identified in the outer membrane sample divided by the
number found in the inner membrane sample; P, P value applying to the magnitude of change (Supplemental Tables S3 and S4) 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
[HSP-OM divided by outer membrane]; values reported here are significant according to the criteria detailed in “Results”); GFP Location,
interpretation of subcellular localization resulting from fluorescence microscopy (ÖMtOM refers to a sole mitochondrial outer membrane
localization, while ÖMtOM++ refers to a localization on the mitochondrial outer membrane and other locations); Reference, references to
previously published location information. A single protein, At5g05520.1 (SAM50-2), demonstrated enrichment in the mitochondrial outer
membrane fractions but did not display specific targeting when tagged with GFP; it is included here due to evidence gathered from protease-treated
mitochondria (Fig. 5). ‘ is used to indicate that the ratio cannot be calculated because a null value (0) was obtained and thus the P value cannot be
calculated, as shown by ^.
Raw Counts
AGI No.
Description
Spectral Counting
OM:HSPOM:IM
Ratio
OM:HSP-OM
iTRAQ
P
Ratio
OM:IM
P
GFP
IM/OM Whole/OM HSP-OM/OM Location
At1g04070.1
TOM9-2
3:0:0
‘
^
‘
^
At1g05270.1
41:7:0
5.9
1.50E-05
‘
^
At1g06530.1
At1g24267.1
At1g27390.1
TraB family
protein
Myosin related
Unknown protein
TOM20-2
53:13:0
13:3:0
6:0:0
4
4.3
‘
5.60E-06
0.022
^
‘
‘
‘
^
^
^
At1g49410.1
TOM6
11:2:0
5.5
0.027
‘
^
At1g50460.1
At1g53000.1
At2g01460.1
41:22:0
53:24:16
8:0:0
1.9
2.2
‘
0.018
0.001
^
‘
3.3
‘
^
0.00003
^
At2g19080.1
HXKL-1
KDSB
ATP
binding/kinase
Metaxin
14:6:1
2.3
0.082
14
0.011
At2g19860.1
At2g38280.1
At2g38670.1
At3g01280.1
HXK2
FAC1
PECT1
VDAC1
92:71:25
7:2:0
55:28:0
90:50:53
1.3
3.5
2
1.8
0.101
0.118
0.004
0.001
3.7
‘
‘
1.7
8.00E-09
^
^
0.002
0.276
0.255
0.264
0.233
0.27
0.254
0.417
0.31
At3g11070.1
At3g20000.1
SAM50-1
TOM40-1
53:24:3
93:42:12
2.2
2.2
0.001
1.90E-05
17.7
7.7
1.30E-06
2.4E-11
0.564
0.217
0.511
0.274
0.429
0.395
At3g20040.1
At3g27080.1
HXK4
TOM20-3
13:4:0
23:0:0
3.2
‘
0.039
^
‘
‘
^
^
At3g27930.1
44:13:0
3.4
0.0001
‘
^
29:13:0
10:4:0
30:5:0
2.2
2.5
6
0.016
0.121
0.0002
‘
‘
‘
^
^
^
At3g63150.1
At4g29130.1
At5g05520.1
At5g08040.1
Unknown
protein
BCS-1
BCS-1-like
Unknown
protein
MIRO2
HXK1
SAM50-2
TOM5
7:0:0
124:97:31
32:8:13
21:12:8
‘
1.3
4
1.75
^
0.07
0.0004
0.122
‘
4
2.5
2.6
^
1.2E-12
0.006
0.02
At5g09420.1
OM64
34:13:0
2.6
0.003
‘
^
0.203
0.211
0.195
At5g12290.1
At5g15090.1
At5g17770.1
72:23:3
97:63:63
34:7:0
3.2
1.5
4.9
1.9E-06
0.008
0.0001
24
1.5
‘
7E-08
0.008
^
0.201
0.422
0.185
0.377
0.446
0.234
0.372
At5g22350.1
DGS1
VDAC3
NADH:cytochrome
B5 reductase
ELM1
17:6:0
2.8
0.028
‘
^
At5g27540.1
At5g40930.1
MIRO1
TOM20-4
89:55:8
8:0:0
1.6
‘
0.005
^
11.1
‘
7E-11
^
At5g43970.1
TOM9-1
15:5:4
3
0.033
3.75
0.019
At3g50930.1
At3g50940.1
At3g58840.1
Reference
Werhahn
et al. (2001)
ÖMtOM
0.285
0.462
ÖMtOM
ÖMtOM
Werhahn
et al. (2001)
Werhahn
et al. (2001)
0.305
ÖMtOM
ÖMtOM
ÖMtOM
0.165
0.229
Lister
et al. (2007)
ÖMtOM
ÖMtOM
ÖMtOM
Lee
et al. (2009)
ÖMtOM
Werhahn
et al. (2001)
ÖMtOM
Werhahn
et al. (2001)
0.162
ÖMtOM
0.231
ÖMtOM
ÖMtOM
ÖMtOM
0.213
0.23
ÖMtOM
ÖMtOM
See legend
Werhahn
et al. (2001)
Chew
et al. (2004)
Xu et al. (2008)
Lee et al. (2009)
ÖMtOM
ÖMtOM
0.2
Arimura
et al. (2008)
0.214
Werhahn
et al. (2001)
Werhahn
et al. (2001)
(Table continues on following page.)
Plant Physiol. Vol. 157, 2011
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Duncan et al.
Table I. (Continued from previous page.)
Raw Counts
AGI No.
At5g55610.1
Description
Unknown
protein
VDAC4
Anion transport
VDAC2
At5g57490.1
At5g60730.1
At5g67500.1
Outer membrane
and other
At5g16870.1 Aminoacyl-tRNA
hydrolase
At5g20520.1 WAV2
At4g35970.1 APX5
At5g39410.1 Unknown
protein
Spectral Counting
iTRAQ
GFP
IM/OM Whole/OM HSP-OM/OM Location
OM:HSPOM:IM
Ratio
OM:HSP-OM
P
Ratio
OM:IM
P
50:19:0
2.6
0.0003
‘
^
80:36:37
13:5:0
122:77:64
2.2
2.6
1.6
0.00007
0.07
0.002
2.2
‘
1.9
0.0001
^
0.00003
16:6:0
2.7
0.04
‘
^
ÖMtOM++
16:3:0
10:4:0
52:34:3
5.3
2.5
1.5
0.008
0.121
0.054
‘
‘
17.3
^
^
1.5E-06
ÖMtOM++
ÖMtOM++
ÖMtOM++
2005). A variety of other proteins in the set of 64 are
known to be proteins in the inner mitochondrial
membrane: 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 Frostbite1 (FRO1; At5g67590.1), unknown protein (At1g68680.1), and unknown protein
(At4g16450.1) are subunits of complex I (Meyer et al.,
2008). Isocitrate Dehydrogenase1 (IDH1; At4g35260.1)
has also been shown to be present in the inner membrane/matrix fraction of mitochondria (Zhao and
McAlister-Henn, 1996; Table II).
MS-iTRAQ
Initial inspection of the putative proteome indicated
the presence of soluble contaminants arising from the
mitochondrial matrix. IDH6 (At3g09810.1) and Aspartate Aminotransferase (ASP1; At2g30970.1; Table II)
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 composed 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 cutoff on the basis that known outer membrane
proteins were included, while known contaminants
were excluded. The validity of this cutoff was confirmed by GFP localization of IDH6 (iTRAQ [for
isobaric tags for relative and absolute quantitation]
mitoplast:outer membrane ratio of 0.8, geometric SD of
1.176) and g-aminobutyrate (GABA) aminotransferase
(At3g22200.1; iTRAQ mitoplast:outer membrane ratio
of 0.776, geometric SD of 1.185), two proteins that fell
close to but outside the cutoff and were shown to
localize to the mitochondria but not to the outer
membrane (Supplemental Fig. S3, I and II). Thus, the
iTRAQ analysis using these criteria contributed evi-
Reference
ÖMtOM
0.187
0.249
0.199
Lee et al. (2009)
ÖMtOM
Lee et al. (2009)
0.464
dence for the removal of four proteins from the list
of 64, namely ASP1, IDH6, GABA aminotransferase,
and a putative NADH:cytochrome B5 reductase
(At5g20080.1).
GFP Tagging
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 (Fig. 4). For
these purposes, an inner membrane protein, FRO1
(At5g67590.1; Heazlewood et al., 2003), a matrix protein, IDH6 (At3g09810.1; Heazlewood et al., 2004),
and a previously characterized outer membrane protein, Elongated Mitochondria1 (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
(Fig. 4). A clear difference was observed between the
fluorescence patterns of the control proteins located in
the inner membrane-FRO1 (Fig. 4A) and matrix-IDH6
(Fig. 4B) and the outer membrane-ELM1 (Fig. 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 (Fig. 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 control (Supplemental Fig.
1100
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
At5g67590.1
At3g09810.1
At3g22200.1
At5g15640.1
At4g04180.1
At1g61570.1
At1g68680.1
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
At4g00355.1
Not mitochondrial
Mitochondrial
At3g63520.1
Not mitochondrial
At4g27760.1
At2g45870.1
Not mitochondrial
At4g33360.1
At2g26240.1
Not mitochondrial
Not mitochondrial
At1g75200.1
Not mitochondrial
Not mitochondrial
At2g30970.1
At5g20080.1
AGI No.
Mitochondrial
Mitochondrial
Location
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Unknown
protein
TIM13
Mitochondrial
substrate
carrier family
protein
Unknown
protein
IDH6
GABA
aminotransferase
Terpene
cyclase/mutaserelated
FRO1
Unknown
protein
FEY
ASP1
NADH-cytochrome
B5 reductase,
putative
Flavodoxin
family
protein
Unknown
protein
Bestrophinlike
protein
CCD1
Description
^
‘
8:0:0
14:3:0
10:0:0
^
^
‘
7:0:0
‘
^
0.00004
‘
3.6
17:0:4
47:13:29
0.015
^
‘
5:0:0
4.7
^
^
10:0:0
4:0:0
‘
^
‘
‘
8:0:0
0.011
2
^
‘
4:0:0
40:20:2
^
‘
9:0:0
^
0.002
‘
3.3
0.008
P
Ratio OM:HSP-OM
^
^
‘
‘
^
^
0.009
0.041
^
^
^
^
3.5E-05
^
‘
‘
4.2
1.6
‘
‘
‘
‘
20
‘
^
^
‘
‘
0.017
0.00015
P
2.6
7.5
Ratio OM:IM
Spectral Counting
3.1
22:7:0
23:0:9
30:9:4
OM:HSP-OM:
IM
Raw Counts
0.557
0.341
1.229
0.713
IM/
OM
0.802
0.776
1.284
Whole/
OM
iTRAQ
0.403
0.51
0.611
CON/
OM
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Mitochondrial
Not
mitochondrial
Not
mitochondrial
Not
mitochondrial
Not
mitochondrial
Not
mitochondrial
Not
mitochondrial
Not
mitochondrial
GFP
Location
Inner
membrane
or matrix
Inner
membrane
or matrix
Inner
membrane
or matrix
Inner
membrane,
complex I
Intermembrane
space
Inner
membrane,
complex I
Previous
Published
Location
Lister
et al.
(2004)
Meyer
et al.
(2008)
Meyer
et al.
(2008)
Reference
(Table continues on following page.)
Inner membrane or matrix
In Vitro
Import Result
Table II. 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 as follows: Location, the subcellular compartmentalization interpretation; AGI No., Arabidopsis Genome Initiative identifier (www.Arabidopsis.org); Description,
common identifier; Raw Counts, tally of the total number of identifying ions summed from three biological replicates from the outer membrane sample followed by the HSP-OM and then the
inner membrane sample; Ratio OM:HSP-OM, number of identifying ions identified in the outer membrane sample divided by the number found in the HSP-OM sample; P, P value applying to
the magnitude of change (Supplemental Tables S1 and S2) as calculated using Poisson regression analysis; Ratio OM:IM, number of identifying ions identified in the outer membrane sample
divided by the number found in the inner membrane sample; P, P value applying to the magnitude of change (Supplemental Tables S1 and S2) 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 [HSP-OM divided by outer membrane]; values reported here are significant according to the criteria detailed in
“Results”); GFP Location, interpretation of subcellular localization resulting from fluorescence microscopy; In Vitro Import Result, mitochondrial localization of tested proteins as determined by
reliance on the presence of an inner membrane electrochemical gradient; Previous Published Location, location of previously studied proteins; Reference, references to previously published
location information. The final protein in this table, 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. ‘ is used to indicate that the ratio cannot be calculated because a null value (0) was obtained and thus the
P value cannot be calculated, as shown by ^.
Arabidopsis Mitochondrial Outer Membrane
1101
Inner
membrane,
complex III
Inner
membrane,
complex I
Inner
membrane,
complex V
Mitochondrial
matrix
Lister
et al.
(2004)
Meyer
et al.
(2008)
Klodmann
et al.
(2010)
Meyer
et al.
(2008)
Zhao and
McAlisterHenn
(1996)
Intermembrane
space
0.32
0.128
6.22E27
10.5
1.5E-07
2.1
220:104:21
Unknown
protein
AtMg01080.1 Subunit 9 of
mitochondrial
F0-ATPase
At4g35260.1 IDH1
Mitochondrial
Mitochondrial
Unknown
protein
At4g16450.1
Mitochondrial
No further evidence At4g17140.2
^
‘
24:0:6
4
^
^
‘
4:0:0
‘
^
^
‘
5:0:0
‘
^
^
UQCRX-like
family protein
At3g52730.1
Mitochondrial
8:0:0
‘
‘
^
^
TIM9
At3g46560.1
Mitochondrial
4:0:0
‘
‘
P
Ratio OM:IM
P
Ratio OM:HSP-OM
Spectral Counting
Raw Counts
OM:HSP-OM:
IM
Description
AGI No.
Location
Table II. (Continued from previous page.)
0.002
0.486
0.203
Whole/
OM
IM/
OM
iTRAQ
CON/
OM
See
legend
GFP
Location
In Vitro
Import Result
Previous
Published
Location
Reference
Duncan et al.
S1), indicating an exclusive mitochondrial outer membrane localization, while four 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 (Supplemental Fig. S2). GFP analysis of the remaining 12
proteins showed that seven of these did not localize to the mitochondria (Supplemental Fig. S4), two
were seen to be mitochondrial but did not display
the ring structure characteristic of outer membrane
proteins (At5g15640.1 and At4g04180.1; Supplemental Fig. S3, III and IV), and three 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 Supplemental
Figures S1 to S4.
In Vitro Uptake Assays
The two mitochondria-localized proteins that did
not display the ring structure (At5g15640.1 and
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 (Supplemental Fig. S3, III
and IV). When valinomycin was 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 localization (Supplemental Fig. S3, 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-kD) protein were fused to GFP but failed to
show a consistent subcellular 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, 3-deoxy-manno-octulosonate cytidylyltransferase (KDSB; At1g53000.1), phosphorylethanolamine
1102
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Arabidopsis Mitochondrial Outer Membrane
cytidylyltransferase (PECT1; At2g38670.1), and the
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 that were protected by the lipid bilayer of the
mitochondrial outer membrane. Treated mitochondria
were separated by SDS-PAGE, and a number of proteins were immunodetected. TOM20-3 is a mitochondrial outer membrane protein that is exposed to the
cytosol (Heins and Schmitz, 1996; Fig. 5A). This protein is rapidly degraded at low concentrations of
proteinase K. Examination of KDSB (Fig. 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 (Fig. 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 S. cerevisiae (Kozjak et al., 2003).
SAM50, like TOM40, is a transmembrane b-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 (Fig. 5, D 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; Fig. 5F), which is
located on the inner mitochondrial membrane, was
resistant to digestion. This indicates that SAM50, in
agreement with the other data presented here, is likely
Figure 4. GFP tagging of mitochondrial outer membrane proteins to
confirm intraorganelle localization.
Arabidopsis suspension cell culture
was transiently transformed with mitochondrial red fluorescent protein (RFP)
control and GFP-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 a similar fluorescence
pattern to ELM1, identified by MS and
location confirmed by fluorescence
microscopy. E, GFP only.
Plant Physiol. Vol. 157, 2011
1103
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Duncan et al.
observed cells (Fig. 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-Lipid A, and has been located
in mitochondria, but not to the outer membrane, in
previous studies in Arabidopsis (Heazlewood et al.,
2004). PECT1 is the penultimate enzyme involved in
the biosynthesis of phosphatidylethanolamine, an important membrane phospholipid. The overexpression
of the outer membrane-targeted fusion protein (Fig.
6B) apparently causes mitochondria to bunch together
at one pole of the cell. Overexpression of a mitochondrial outer membrane NADH:cytochrome B5 reductase (Fig. 6C) also appears to alter mitochondrial
morphology when compared with the more typical
mitochondrial outer membrane fluorescence pattern
seen with the unknown protein At5g55610.1 (Fig. 6D).
Transcriptomic Analysis of Genes Encoding Outer
Membrane Proteins Reveals Patterns of Coexpression
Figure 5. Immunodetection of isolated mitochondria treated with
proteinase K. A, TOM20-3 is a mitochondrial outer membraneanchored protein with a large cytosolic domain and as such is
vulnerable to digestion by external application of proteinase K. B and
C, KDSB (B) and PECT1 (C) are novel outer membrane proteins
identified in the course of this study by MS and confirmed by GFP
localization. The breakdown of KDSB is similar to that of TOM20-3,
indicating that it is also exposed to the cytoplasm. PECT1 is broken
down by external application of proteinase K but appears to be more
resistant than TOM20 and KDSB. D and E, SAM50 (D) and TOM40 (E)
are b-barrel outer membrane proteins and as such are embedded in the
membrane, rendering them resistant to digestion. F, RISP is found in the
ubiquinol-cytochrome c reductase complex in the mitochondrial inner
membrane. Its preservation indicates that the integrity of the inner
membrane was maintained throughout the proteinase dilution series.
to be located on the outer mitochondrial membrane in
Arabidopsis and that mitochondrial integrity was not
compromised by the proteinase treatment.
Transient Overexpression of Outer Membrane Protein
Affects Mitochondrial Morphology and Segregation
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 KDSB (At1g53000.1), highly abnormal giant
mitochondria occupied much of the available space in
In order to determine whether there was a relationship between colocalization and coexpression, 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 subsets of outer membrane proteins, differed from the patterns of transcript abundance for
genes that encode inner membrane proteins. Data
were normalized to make them comparable (see
“Materials and Methods”), and expression levels are
displayed relative to maximum expression (Fig. 7;
Supplemental Fig. S5). Overall, it was observed that
the 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
(Supplemental Fig. S5). When all genes encoding inner
membrane- and outer membrane-localized proteins
were clustered together over a germination time
course, it was seen that genes encoding outer membrane proteins were significantly underrepresented in
cluster 2 (P , 0.05) compared with genes encoding
inner membrane proteins (13% outer membrane versus 27% inner membrane; Fig. 7). Gene in cluster 2
displayed high transcript abundance in fresh harvested seeds that decreased during stratification. In contrast, 45% of genes encoding outer membrane proteins
(versus 21% of inner membrane protein-encoding
genes) were seen in the group of genes showing
transient expression during germination in cluster 4
(Fig. 7, boxed in yellow), which represents a significant
overrepresentation (P = 0.0007) of genes encoding
outer membrane proteins in this cluster. Genes in
cluster 4 peak in transcript abundance during the first
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Arabidopsis Mitochondrial Outer Membrane
Figure 6. Transient expression of several fluorescently tagged fusion proteins causes apparent alterations in
mitochondrial morphology/distribution.
A, 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, PECT-1. The mitochondria bunch
tightly together at one pole of the cell.
C, NADH:cytochrome B5 reductase.
The mitochondrial population has an
abnormal size distribution with many
large mitochondria. D, Unknown protein, commonly observed mitochondrial morphology for comparison.
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 radicle
has emerged (Weitbrecht et al., 2011). Thus, although
genes encoding mitochondrial outer membrane proteins follow the general pattern 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 subset (i.e. cluster 4) this differs
from the majority of genes encoding mitochondrial
proteins. Previously, it has been shown for maize (Zea
mays) and rice (Oryza sativa) that active mitochondrial
biogenesis precedes the expression of respiratory
chain components of the inner membrane (Logan
et al., 2001; Howell et al., 2006); thus, the pattern
observed here suggests that the establishment 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,
coordinated cellular processes such as apoptosis
(Lindsay et al., 2011), organelle division (Kuroiwa,
2010), mitochondrial inheritance (Koshiba et al.,
2011a), 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 subcellular
localizations; the identification of expected, but as yet
uncharacterized, protein components of biological pro-
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Duncan et al.
Figure 7. Coexpression of genes encoding outer mitochondrial membrane proteins. Publicly available microarrays analyzing
wild-type tissues in the AtGenExpress developmental set (Schmid et al., 2005; E-AFMX-9) and during germination show the
highest expression of genes encoding inner membrane and outer membrane proteins during germination (Supplemental Fig. S5).
The expression of genes encoding outer membrane proteins was analyzed during germination. Har, Seeds harvested on the day of
collection from wild-type plants; 0 h, seeds 15 d after ripening, prior to imbibition; S, hours in cold stratification in the dark; SL,
hours into continuous light after 48 h of stratification. Expression levels were made relative to maximum expression,
hierarchically clustered (Euclidean distance and average linkage clustering), and five clusters were observed (C1–C5). A group of
genes showing transient expression are indicated in the yellow box. Details for each of the tissues analyzed by microarrays are
shown in Supplemental Table S6.
cesses; and as the basis for launching the investigation
of novel, uncharacterized protein components, with the
potential to lead to the assignment of new and unexpected biological functions. The analysis of the mitochondrial outer membrane in plants is especially
interesting because the presence of the chloroplast in
plant 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 copurify multiple structures
at the same time. This is a recurring theme in subcellular proteomics, especially in subcellular membrane
proteomics (Lilley and Dunkley, 2008). Combining
methods that 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 provides a solid basis for determining subcellular and suborganelle localization.
In combination, this is a highly complex and timeconsuming 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 the
enrichment of known and unknown proteins between
samples. However, we have found that the Exponentially Modified Protein Abundance Index and the
Normalized Spectral Abundance Factor for quantita-
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Arabidopsis Mitochondrial Outer Membrane
tive 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
the 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
that are not detected in more crude samples. These
problems affect the statistical analysis of both spectral
counting and iTRAQ data, as data sets are not normally distributed in the cases of the most enriched and
interesting proteins from the perspective of subcellular
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
the analysis of membrane systems of low abundance
(Fig. 1).
There are three key aspects of this workflow. First,
selection of multiple independent prefractionated
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 the
coenrichment of contaminating structures in the Suc
gradients (HSP-OM), and a control for nonmembrane
proteins associating with the membrane during the
cell fraction procedures (mitoplasts). Second, inclusion
of proteins observed in enriched fractions and absent
from prefractionated samples. Such proteins should be
included despite issues related to the normality of
distributions and limited spectral counts, as long as
the spectra are sufficient to prove identification, as
these include low-abundance proteins only found
through enrichment. Third, multiple confirmation
strategies that are independent of the enriched fractions are very helpful, especially those that relate to
targeting of proteins in situ and/or in vitro to the
location identified. In this study, we used a diverse
literature of counterclaims, GFP labeling in intact cells,
and in vitro protein import. In addition to this, proteinprotein 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 I). 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 VDACs, specifically, the five known import
receptors (TOM 20-2, 20-3, 20-4, OM64, and Metaxin)
and components of the central import pore (TOM 40,
9-1, 9-2, 5, and 6) and VDAC1 to -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 expected, but not
detected, in this study were TOM7 (Werhahn et al.,
2001) and BIGYIN, the Arabidopsis ortholog to
S. cerevisiae and human Fis1 (Scott et al., 2006).
In addition to the 16 known plant outer mitochondrial membrane proteins, four 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 and At5g05520.1), which are evolutionarily conserved across kingdoms, and orthologs 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 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 S. cerevisiae (Frederick et al., 2004). Thus, experimentally confirming the localization of these in plants,
to our knowledge 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 knockouts 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 to our
knowledge for the first time in this study. Examination
of these revealed the presence of Hexokinase1 (HXK1;
At4g29130.1), HXK2 (At2g19860.1), and HexokinaseLike Protein1 (HXKL1; At1g50460.1) on the mitochondrial outer membrane (Table I). The Arabidopsis
hexokinases have long been reported to be associated
with mitochondria, first in 1983 (Dry et al., 1983;
Damari-Weissler 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 transmembrane region of 20 to 25 amino
acids, high hydrophobicity at the N terminus (Supplemental Fig. S1, IV, VII, and XVII), the ability to
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Duncan et al.
target GFP to mitochondria, combined with its enrichment in the outer membrane samples (Table I) and a
lack of detection of other glycolytic enzymes suggests
that these proteins are authentic outer membrane
proteins. The location of these hexokinases 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 hexokinases 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-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, a NADH:cytochrome B5 reductase
(At5g17770.1) might be expected to be in the outer
membrane of mitochondria, based on the localization
of its S. 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 membranebound 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 on the endoplasmic reticulum, as assumed
previously.
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 b-Barrel Protein
One of the proteins identified in the outer membrane proteome was a novel b-barrel protein
(At3g27930.1), bringing the total number of distinct
b-barrel proteins identified in the Arabidopsis mitochondrial outer membrane to four (TOM40, SAM50,
VDAC, and At3g27930.1; Table I). Amino acid sequence alignments comparing this novel protein with
other membrane b-barrel proteins (TOM40, SAM50,
VDAC, MDM10, OMP85, and TOC75) from diverse
species revealed that this b-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 TAIR 10), upon searching
the literature, it was found that this gene has been
identified as on the list of 437 proteins making 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
At3g27930.1 on this list. Taken together with the confirmed outer mitochondrial membrane localization
(Table I), high expression in seed and early germination (Fig. 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 a
novel import component.
Proteins Involved in the 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-like domains and is predicted to catalyze the conversion of
uridine monophosphate/cytidine monophosphate
to their diphosphate forms. It has previously been
demonstrated that a nucleoside diphosphate kinase
(At4g11010.1) is present in the mitochondrial intermembrane 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
et al., 2006). This enzyme catalyzes the transfer of
phosphoethanolamine onto cytidine triphosphate +
inorganic pyrophosphate to yield cytidine diphosphoethanolamine (CDP-ethanolamine). The final
enzyme required for the production of phosphatidyl-
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Arabidopsis Mitochondrial Outer Membrane
ethanolamine is CDP-ethanolamine phosphotransferase, the location of which is not yet characterized in
Arabidopsis but is found in the endoplasmic reticulum
in S. cerevisiae (Jelsema and Morré, 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
promoter, resulting in transient overexpression 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 (Supplemental Fig. S1, IX), suggesting that PECT1 is not
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 3-deoxy-D-manno-2octulosonic acid (Kdo) transferase, which catalyzes
the transfer of Kdo onto cytidine triphosphate to form
CMP-Kdo (Séveno et al., 2010). This protein is orthologous to the Escherichia coli protein KDSB, which
participates in the synthesis of KDO2-Lipid 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 KDO2-Lipid 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 (Fig. 4A) suggests the presence of
KDO2-Lipid A or a related compound in the outer
membrane of mitochondria.
domain in common and lacks the BCS domain. Interestingly, it differs from 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 (Fig. 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., 2011).
AAA-Type ATPases
CONCLUSION
Two related proteins containing ATPases associated
with diverse cellular activities (AAA)-type ATPase
domains were identified on the outer membrane,
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 synthesis (BCS)-like protein, closer examination reveals that it only contains the AAA-ATPase
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. Dualtargeted or multitargeted 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 addi-
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 reactive oxygen species levels and plant
growth, respectively (Karve and Moore, 2009; BolouriMoghaddam et al., 2010), while MIRO proteins have
been observed in regulating mitochondrial transport
and morphology (Yamaoka and Leaver, 2008; Reis
et al., 2009). Another protein that may 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, its role in
eukaryotic cells is still unknown. Nevertheless, the
identification of this protein implies a conserved role
for it, both on bacterial membranes and evidently on
the mitochondrial outer membrane in plants. Another
well-conserved protein is Embryonic Factor1 (FAC1;
At2g38280.1), for which the subcellular 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.
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Duncan et al.
tional components of the SAM complex of the outer
membrane were identified that might be expected to
be present, in comparison with the SAM complex
from yeast. Nevertheless, 27 proteins were experimentally determined to be located on the outer mitochondrial membrane, to our knowledge 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 S. 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, to our knowledge, the first
functionally related information for the proteins
encoded at these loci. Examination of the full list of
proteins identified (Table I) 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-like proteins, which play roles in
cell death in mammalian systems (Lindsay et al.,
2011), the GTP protein b2 subunit, which plays a role
in regulating mitochondrial fusion in mammalian
systems (Zhang et al., 2010), Mim1, which 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 at
least some of the proteins that we identified with
no known functions may fulfill similar roles. These
findings, together with the identification of five
plant-specific genes (Plant Specific Protein Database;
Gutiérrez et al., 2004), including a novel b-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.
MATERIALS AND METHODS
Purification of Mitochondria from Arabidopsis
Suspension Cell Culture
Protoplasts were prepared from 6-d-old Arabidopsis (Arabidopsis thaliana) suspension cell cultures. Typically, 500 g fresh weight of cells was
collected by filtration and incubated for 3 h at 21°C with shaking at 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). Protoplasts were collected by centrifugation at 800g for 10 min, resuspended in 500 mL of 0.4 M Suc, 50 mM Tris, 3 mM
EDTA, 20 mM Cys, 0.1% (w/v) bovine serum albumin (BSA), pH 7.5, and
HCl and ruptured in a Potter-Elvehjem homogenizer. Cell debris was
removed by centrifugation at 2,500g for 5 min, and the organellar fraction
(supernatant) was pelleted at 20,000g for 20 min. A portion (1/16th) of this
material was removed and stored at 4°C for preparation of the HSP and
HSP-OM samples. 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 at 40,000g for 40 min.
The 25%/40% (v/v) interphase was removed and added to 300 mL of 0.3 M
Suc, 10 mM TES, and 0.2% (w/v) BSA, pH 7.5 (Suc wash buffer), and
centrifuged at 20,000g for 15 min. Subsequently, the supernatant was
removed and the pellet was layered onto 40% (v/v) Percoll in Suc wash
buffer and centrifuged at 40,000g for 40 min. The gradient-purified mitochondria were removed and washed twice in Suc wash buffer. A total of 500
mg of this sample was removed and stored at 4°C for later analysis.
Typically, 100 to 150 mg of mitochondrial protein (Bradford assay) was
recovered.
The gradient-purified mitochondria were further purified by FFE 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-mm membrane
filter (Bio-Rad) and then passed through a 5-mm 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 at 20,000g for 10 min and washed in Suc wash medium 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 HSP 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 Suc gradient. An inner membrane fraction was
prepared by diluting this in 11 mL of Suc wash medium before freezing/
thawing five times and pelleting at 504,000g 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 freezing/
thawing procedure was included in the iTRAQ experiment.
Western-Blot Analysis
Ten micrograms of membrane fractions and 20 mg of organelle fractions
per lane were separated by 14% (w/v) SDS-PAGE. Proteins were transferred
onto nitrocellulose membranes. Primary antibodies were as follows:
TOM40-1 (At3g20000.1) at 1:5,000 for 1 h at 21°C and developed according
to the manufacturer’s instructions (Roche; Carrie et al., 2009); KAT2 antibody (Germain et al., 2001) was used at 1:1,000 for 1 h at 21°C; Rubisco large
subunit and Calreticulin antibodies were obtained from Abcam and used at
1:500 for 18 h at 4°C; E1a antibody was obtained from Tom Elthon
(University of Nebraska) and used at 1:2,000 for 1 h at 21°C; COXII antibody
was obtained from Agrisera and used at 1:5,000 for 1 h at 21°C. Antibodies to
TOM20-3 (Lister et al., 2007), KDSB, SAM50, PECT1, and RISP were created
by purifying 63His-tagged recombinant proteins from Escherichia coli
expression using Profinia-based immobilized metal affinity chromatography (Bio-Rad). The purified proteins were used to generate rabbit polyclonal antibodies.
Proteinase K Titrations
A total of 250 mg of isolated mitochondria was resuspended in 0, 2, 8, 32,
and 64 mg mL21 proteinase K (Sigma-Aldrich) in 2.5 mL of 0.4 M Suc, 50 mM
Tris, 3 mM EDTA, 0.1% (w/v) BSA, pH 7.5, and HCl. Samples were incubated
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Arabidopsis Mitochondrial Outer Membrane
on ice for 30 min before dilution in 30 mL of 0.4 M Suc, 50 mM Tris, 3 mM EDTA,
0.1% (w/v) BSA, pH 7.5, and HCl containing 1 mmol of Pefabloc SC (Roche).
Samples were centrifuged at 20,000g for 15 min, resuspended in Laemmli
sample buffer, and separated by SDS-PAGE.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the accession numbers listed in Supplemental Table S8.
Supplemental Data
MS
The following materials are available in the online version of this article.
Label-Free Analysis and Statistical Assessment
Supplemental Figure S1. Members of the mitochondrial outer membrane
proteome, as shown by fluorescence microscopy of transiently transformed Arabidopsis cell culture or onion (Allium cepa) cells with fulllength coding sequences fused to GFP.
Fifty micrograms of outer membrane, HSP-OM, 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 at 20,000g for 5 min. Samples were then dried
down in a vacuum centrifuge and analyzed on an Agilent 6510 Q-TOF
mass spectrometer according to a modification of the methods reported by
Eubel et al. (2008), as outlined in detail in Supplemental Materials and
Methods S1. Spectra are available via the ProteomeCommons Tranche Project
hash: 3y16ynJWUCAiKGSfZNprN/paRI32nQA9XM0asqZItzPwnf2jEB/
zsnOAoSxno14kymUwnJbylKkk4cA2sGdLN52sAQ0AAAAAAAAHqg = =.
Peptide count data were compiled from three biological replicates each of
the outer membrane, HSP-OM, and mitochondrial inner membrane samples,
as outlined in detail in Supplemental Materials and Methods S1.
iTRAQ Labeling and Data and Statistical Analysis
Supplemental Figure S2. Members of the mitochondrial outer membrane
proteome that are also found in other subcellular locations, as shown by
fluorescence microscopy of transiently transformed Arabidopsis cell
culture or onion cells with full-length coding sequences fused to GFP.
Supplemental Figure S3. Members of the putative mitochondrial outer
membrane proteome that have been shown to be mitochondrial but not
outer membrane by fluorescence microscopy of transiently transformed
Arabidopsis cell culture or onion cells with full-length coding sequences
fused to GFP.
Supplemental Figure S4. Members of the putative mitochondrial outer
membrane proteome that have been shown not to be mitochondrial by
fluorescence microscopy of transiently transformed Arabidopsis cell
culture or onion cells with full-length coding sequences fused to GFP.
A total of 100 mg of four samples (outer membrane [Mt OM], contaminant
outer membrane [HSP-OM], mitoplast, and inner membrane [IM]), each
created by pooling three biological replicates, were analyzed according to a
modification of the method reported previously (Shingaki-Wells et al., 2011) as
outlined in Supplemental Materials and Methods S1. Quantitation was carried
out using an in-house quantitation method that carried out protein identifications and quantitation on isobaric tags of mass 114 to 117 at the peptide
level. Ratios for individual peptide matches were obtained from peptides
meeting the minimum criteria outlined above and were then combined to
determine ratios for protein 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 Supplemental Materials and Methods S1.
Supplemental Figure S5. Coexpression of genes encoding outer mitochondrial membrane proteins.
In Vitro Imports
Supplemental Table S5. Details of the germination microarrays and
developmental tissue set (Schmid et al., 2005; E-AFMX-9).
In vitro import of putative outer membrane proteins into isolated mitochondria was performed as described previously (Lister et al., 2007). Data are
provided in Supplemental Figure S3, II, III, and IV.
Supplemental Table S6. Details of the genes defined as encoding inner
mitochondrial membrane proteins.
Supplemental Table S1. List of the 185 proteins detected in two or more of
the outer membrane-enriched biological replicates (www.Arabidopsis.
org).
Supplemental Table S2. List of the 64 members of the putative outer
membrane proteome with evidence for identification and enrichment in
the outer membrane fraction (www.Arabidopsis.org).
Supplemental Table S3. Summary of the location evidence gathered in the
course of this research.
Supplemental Table S4. Summary of data gathered from iTRAQ analysis
of the outer membrane-enriched and three prefractionation samples.
Supplemental Table S7. Details of polypeptide identifications resulting
from the in-gel digestion of bands excised from the gel pictured in
Figure 3A
GFP 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 cotransformed into Arabidopsis suspension cell culture with a red
fluorescent protein control by particle bombardment as outlined previously
(Carrie et al., 2009). Data are provided in Supplemental Figures S1 to S4.
Supplemental Table S8. Sequence data.
Supplemental Materials and Methods S1. Detailed MS methods.
Received July 9, 2011; accepted August 31, 2011; published September 6, 2011.
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