Download The potato tuber mitochondrial proteome

Plant Physiology Preview. Published on December 18, 2013, as DOI:10.1104/pp.113.229054
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Running head: The potato tuber mitochondrial proteome
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Corresponding author:
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Dr. Ian Max Møller
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Department of Molecular Biology and Genetics
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Science and Technology
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Aarhus University
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Forsøgsvej 1
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DK-4200 Slagelse
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Denmark
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E-mail address:
[email protected]
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Research area: Cell Biology
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Title: The potato tuber mitochondrial proteome
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Salvato, Fernandaa,e), Havelund, Jesper F.b,c) , Chen, Mingjiea), Rao, R.S.P.a), Rogowska-
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Wrzesinska, Adelinac), Jensen, Ole N.c), Gang, David R.d), Thelen, Jay J.a) and Møller,
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Ian Maxb)
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a) Department of Biochemistry and Interdisciplinary Plant Group, University of
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Missouri-Columbia, Christopher S. Bond Life Sciences Center, Columbia, MO 65211,
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USA
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b) Department of Molecular Biology and Genetics, Science and Technology, Aarhus
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University, Forsøgsvej 1, DK-4200 Slagelse, Denmark
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c) Department of Biochemistry and Molecular Biology, University of Southern
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Denmark, Campusvej 55, DK-5230 Odense M, Denmark
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d) Institute of Biological Chemistry, Washington State University, Pullman,
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Washington 99164 USA
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e) Present address: Institute of Biology, State University of Campinas, São Paulo, Brazil
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One-sentence summary:
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A high-coverage potato tuber mitochondrial proteome uncovers many new proteins and
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functions, especially in coenzyme and iron metabolism, and many posttranslational
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modifications
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Footnote:
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This study was supported by grants from the Danish Council for Independent Research
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– Natural Sciences (to IMM) and a 2012 Sabbatical fellowship (to JJT) from the OECD
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Co-operative Research Programme: Biological Resource Management for Sustainable
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Agricultural Systems.
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ABSTRACT
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Mitochondria are called the powerhouses of the cell. To better understand the role of
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mitochondria in maintaining and regulating metabolism in storage tissues, highly
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purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum L.
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cv. Folva) and their proteome investigated. Proteins were resolved by one-dimensional
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gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by
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liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four
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different search programs, a total of 1060 non-redundant proteins were identified in a
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quantitative manner using normalized spectral counts including as many as five-fold
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more “extreme” proteins (low mass, high pI, hydrophobic) than previous mitochondrial
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proteome studies. We estimate that this compendium of proteins represents a high
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coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The
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dynamic range of protein expression spanned 1800-fold and included nearly all
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components of the electron transport chain, tricarboxylic acid cycle, and protein import
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apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29
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membrane carriers/transporters, a number of new proteins involved in coenzyme
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biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C
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protein phosphatase that may catalyze the dephosphorylation of the pyruvate
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dehydrogenase complex. Systematic analysis of prominent post-translational
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modifications (PTM) revealed that >50% of the identified proteins harbor at least one
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modification. The most prominently observed class of PTMs was oxidative
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modifications. This study reveals approximately 500 new or previously unconfirmed
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plant mitochondrial proteins and outlines a facile strategy for unbiased, near-
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comprehensive identification of mitochondrial proteins and their modified forms.
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INTRODUCTION
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Plant mitochondria participate in a number of processes in the plant cell depending on
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the cell, tissue, or organ type, the developmental stage, and the environmental
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conditions (Rasmusson et al. 2010, Millar et al. 2011). Important examples are energy
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metabolism, photorespiration, amino acid biosynthesis, coenzyme (vitamin)
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biosynthesis, and programmed cell death. All of these processes require that the
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mitochondria can exchange metabolic intermediates and information with the rest of the
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cell via membrane carriers and signaling pathways. Plant mitochondria also import
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>95% of their proteins across the inner and outer mitochondrial membranes (IMM and
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OMM, respectively). Finally, mitochondria are semi-autonomous organelles capable of
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growing and dividing, and as such they perform DNA replication, DNA transcription,
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and protein biosynthesis, in addition to protein import.
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All of these processes require proteins and the composition of the plant mitochondrial
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proteome therefore changes depending on the conditions (Millar et al. 2005). It has been
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estimated that as many 2000-3000 gene products belong to the mitochondria in
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Arabidopsis, but it is not known how many are expressed in a single tissue under a
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specific set of conditions (Millar et al. 2005, Millar et al., 2006, Cui et al., 2011). To
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date the most complete global proteomic study of isolated mitochondria identified 416
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proteins in mitochondria from Arabidopsis cell cultures grown under standard
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conditions with no photosynthesis (Heazlewood et al. 2004). In the intervening years a
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number of more focused proteomic studies have been performed mainly on Arabidopsis
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mitochondria swelling the number of identified plant mitochondrial proteins to 660 non-
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redundant proteins (Heazlewood et al., 2004, Duncan et al., 2011, Klodmann et al.
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2011, Lee et al., 2012, Tan et al., 2012). Missing from this list are many essential plant
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mitochondrial activities including regulatory proteins, transcription factors, metabolite
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translocators, and the wealth of tRNA synthases and pentratricopeptide repeat (PPR)
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proteins (Havelund et al. 2013).
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In yeast (Saccharomyces cerevisiae) about 850 proteins have been identified in isolated
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mitochondria, which is thought to provide 85% coverage of the proteome in this species
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(Premsler et al. 2009). A total of 1117 proteins were identified in mitochondria from the
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nematode Caenorhabditis elegans (Li et al. 2009). The total mammalian mitochondria
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is predicted to contain 1050-1400 proteins (Calvo and Mootha 2010) and a global
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proteomic study of mitochondria isolated from fourteen mouse tissues using one-
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dimensional polyacrylamide gel electrophoresis (1D-PAGE) followed by liquid
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chromatography and two-dimensional mass spectrometry (LC-MS/MS) identified 1098
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different gene products (Pagliarini et al. 2008).
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Considering that the total plant mitochondrial proteome is thought to contain almost
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twice as many proteins as the nematode and mammalian mitochondrial proteomes, it is
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likely that an in-depth proteomic study of one type of plant mitochondria would identify
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more than 1000 proteins or about twice the previous most complete study. It is likely
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that newly discovered mitochondrial proteins would be of low abundance, which
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conceivably would lead to the discovery of new pathways and regulatory proteins, as
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well as providing additional information about established pathways.
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In the present study, we describe the proteome of potato tuber mitochondria (POM).
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The potato tuber is a large, relatively homogeneous storage organ and potato tuber
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mitochondria are therefore expected to be similarly homogeneous. There are well-
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established methods for isolating these mitochondria in a purified, intact and functional
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form (Neuburger et al. 1982, Struglics et al. 1993, Considine et al. 2003) and their basic
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properties are therefore well-characterized. In addition, the potato genome has recently
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been sequenced (Xu et al. 2011), which greatly simplifies the task of identifying potato
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proteins. Using a prefractionation by preparative SDS-PAGE followed by high-mass
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accuracy LC-MS/MS we have achieved a high proteome coverage of 1060 proteins,
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discovered a number of new functions, and identified a large number of
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posttranslational modifications, particularly oxidative modifications.
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RESULTS
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The isolated potato tuber mitochondria are highly purified
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The isolation of pure organellar fractions from crude source material is the critical phase
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of any subproteome analysis. It has previously been established that very pure
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mitochondria can be obtained from potato tubers, which only contain 0.2% peroxisomes
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or plastids on a protein basis (Neuburger et al. 1982, Struglics et al. 1993, Considine et
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al. 2003). We employed this established procedure for the isolation of mitochondria
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with a recognized low contamination level.
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We also tested the mitochondrial purity by Western blotting using subcellular marker
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proteins. The proteins14-3-3 and anti-enolase were used as reference markers for the
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cytosolic compartment, while alpha carboxytransferase (α-CT) and pyruvate
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dehydrogenase E1 component subunit alpha (PDE1-α) were used as markers for the
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plastidic and the mitochondrial compartment, respectively. Anti-PDE1-α demonstrated
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the expected mitochondrial enrichment in the mitochondrial fraction, while there was no
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detectable cytosolic or plastidic contamination (Figure S1).
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The potato tuber mitochondrial proteome contains more than 1000 proteins
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Previous Arabidopsis mitochondrial proteome studies, both shotgun and targeted, have
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collectively identified a set of 660 non-redundant proteins (Heazlewood et al., 2004,
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Duncan et al., 2011, Klodmann et al. 2011, Lee et al., 2012, Tan et al., 2012). These
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studies employed primarily either two-dimensional (2D) gel electrophoresis or gel-free
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analyses followed by tandem mass spectrometry (MS). In the present study we
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performed one-dimensional gel electrophoresis followed by liquid chromatography-
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tandem mass spectrometry (GeLC-LC-MS) to overcome limitations of 2D gels while
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allowing for preparative level proteome interrogation. Using GeLC-MS, proteins from
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each replicate were prefractionated by 12% SDS-PAGE and sectioned into 40 gel slices
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prior to trypsin digestion, effectively reducing sample complexity prior to LC-MS/MS
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(Figure 1). To maximize the number of assignments, four complementary database-
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searching programs were employed simultaneously. Using a false discovery rate (FDR)
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cutoff of 1% at the protein level, this approach resulted in 7346 assigned non-redundant
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peptides representing 1060 protein groups including 23 that are mitochondrially
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encoded. The average coverage was 24.9% (Table I, Table S1).
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Figure 2 shows the overlapped peptides and proteins identified using MSGF-DB,
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ProLuCID, Sequest, and Mascot. A comparison of these different search algorithms
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revealed that MSGF-DB outperformed the other search engines by identifying 85% of
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the total peptides, compared to 76% identified by ProLuCID, 46% by Sequest, and 37%
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by Mascot.
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Evaluation of the mitochondria proteome by transient fluorescence assay in
tobacco epidermal cell
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To evaluate the false positive rate of the mitochondria proteome data, 20 different
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proteins were selected for further evaluation (Table S1, Figure 3, Figure S2). These
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selected proteins either were of relatively low abundance and/or their mitochondrial
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localization was at variance with previous reports. In other words, they were potential
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contaminants. An EYFP fluorescence tag was in frame fused at their C-terminal; each
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construct was mixed with a mitochondrial marker (CD3-986) and co-transformed into
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tobacco leaves for subcellular localization by fluorescence microscope. Out of the total
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20 constructs, 19 were detected in tobacco leaves at variable levels, and 18 of them
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were confirmed to localize to mitochondria. PGSC0003DMP400005581, which encodes
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a putative inorganic pyrophosphatase, was found to localize to chloroplasts (Figure 3).
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Its ortholog in Arabidopsis (At5g09650) was confirmed to localize to chloroplasts
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(Schulze et al., 2004). So this protein is likely to be a contaminant.
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Two proteins (PGSC0003DMP400035788 and PGSC0003DMP400024128) were found
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with dual localization. PGSC0003DMP400035788, annotated as pentatricopeptide
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repeat-containing protein, was found to localize to both mitochondria and chloroplasts
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(Figure S2). Its ortholog in Arabidopsis (At3g46870) was identified as a plastid protein
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by organellar proteomics (Kleffmann et al, 2004); it also was identified as a Zn2+-IMAC
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interaction protein in mitochondria (Tan et al, 2010). Its dual localization confirmation
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here resolved the controversy between these two independent investigations (Kleffmann
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et al, 2004; Tan et al, 2010). PGSC0003DMP400024128, which encodes an RNA-
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binding protein 24-like, was found to localize to mitochondria and another unknown
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organelle (Figure S2). This protein is low abundance and none of the subcellular
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prediction algorithms predicted it to localize to mitochondria.
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Two proteins (PGSC0003DMP400012523 and PGSC0003DMP400050514) were
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observed to localize to mitochondria (Figure S2), and these observations contradict
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previous reports. PGSC0003DMP400012523’s Arabidopsis ortholog (At1g78590) was
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reported to localize to the cytosol although that conclusion is open to question as no
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cytosolic marker was used (Chai et al., 2006). PGSC0003DMP400050514’s
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Arabidopsis ortholog (At2g37250) was reported to locate to chloroplasts (Carrari et al.,
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2005). Since this Arabidopsis ortholog falls into the same phylogenetic group as the
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potato plastidial isoform of adenylate kinase,with which it showed 76% identity, it will
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require additional phylogenetic analysis to determine their orthologs. The existence of a
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mitochondrial isoform of adenylate kinase in potato tuber was first reported by Roberts
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et al. (1997).
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In summary, these EYFP-localization results are consistent with the conclusion that the
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potato tuber mitochondria are highly purified and that no more than 5% of the 1060
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identified mitochondria proteins are false positives. This ratio could be well be
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overestimated since the candidates for the localization study were purposely selected
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based on their low abundance and their contradiction with reported results in the
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literature.
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Categorization of the identified proteins according to properties and function
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Considering proteins only detected in at least two replicates, we have identified 884
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protein groups (Table S3), of which 32% were confirmed by ortholog analysis when
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compared to the Arabidopsis mitochondrial proteome (Heazlewood et al., 2004, Duncan
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et al., 2011, Klodmann et al. 2011, Lee et al., 2012, Tan et al., 2012). Thus 528 new
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non-orthologous proteins are presented in the current study, in comparison to all
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previous Arabidopsis mitochondrial proteome investigations. As shown in Figure 4, the
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potato mitochondrial proteins were distributed between 4 to 161 kDa in size, between -
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0.9 to +0.3 in GRAVY score, and with pI values ranging from 4 to 11. A comparison of
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the physical properties of the potato versus the Arabidopsis mitochondrial proteome
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(Heazlewood et al., 2004) demonstrates that a much larger number of small proteins
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with negative GRAVY scores have been identified in the potato mitochondrial
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proteome (Figure 4).
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Assigned potato mitochondrial proteins were annotated by BLAST querying against the
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non-redundant NCBI database and were classified based on the gene ontology terms
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related to biological process and molecular function (Figure S3). In addition, each
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protein was assigned to thirteen general functional categories according to the
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classification by Heazlewood et al. (2004). The distribution of proteins classified in
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each category is compared between potato and Arabidopsis mitochondrial proteomes
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(Table I). The largest functional classes in the potato mitochondrial proteome were
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energy (12%), metabolism (20%), and protein synthesis (12%). Altogether these
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proteins represent 45% of total protein abundance based on normalized spectral
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counting (dNSAF). These proteins are mainly involved in the tricarboxylic acid cycle
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(TCA), oxidative phosphorylation, and protein synthesis (mainly ribosomal and tRNA
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synthetase proteins). Around 23% of the identified proteins (also, 23% of the
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abundance) were categorized in the RNA processing, DNA synthesis and processing,
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transcription or protein fate classes. In addition, approximately 12% of the identified
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proteins (also 12% of the abundance) were related to transport, or
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defense/detoxification, or signaling processes.
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The number of proteins found in each functional category was higher for potato than for
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Arabidopsis with the exception of structural organization, although the increase for the
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energy category was relatively small (1.3 fold) (Table I). The most marked increase in
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number of proteins identified was seen for RNA processing and protein synthesis where
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7.4-fold and 8.4-fold more proteins were found in potato, but also the category transport
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showed a large increase (3.6 fold). The relative frequency (%) of proteins in each
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category were very similar, with the exception of the energy category, which was
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significantly lower in potato, and RNA processing and protein synthesis categories,
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which were significantly higher. Unclassified proteins, that is, proteins not confidently
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assigned to any functional class, comprised 21% of the total number of identified
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proteins (20% of the abundance) in the potato mitochondrial proteome (Table I).
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A number of new proteins and enzymes were identified that increase our coverage of
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known mitochondrial processes and, more interestingly, extend the list of mitochondrial
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processes for instance with respect to coenzyme and iron metabolism. This will be
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treated in the Discussion.
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The abundance of mitochondrial proteins varies more than 1800-fold
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It has been shown that in LC-MS/MS based analysis of peptide mixtures raw spectral
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counts correlate with relative protein abundance between different samples (Liu et al.,
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2004; Stevenson et al., 2009, Matros et al., 2011). However, to compare the relative
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abundance of different proteins within the same biological sample spectral counts for
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each protein must be normalized. One of the most logical and promising strategies for
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normalization is based on protein length (Zhang et al., 2010).
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In the present work, proteins that were detected in at least two replicates (884 proteins)
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were assigned an abundance factor (Table S3). For this, we calculated the distributed
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normalized spectral abundance factor (dNSAF) which considers unique spectral counts
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and distributes the shared spectral counts matched to peptides assigned to homologous
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proteins. Spectral counts of each protein were then normalized by protein length (see
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Methods for details).
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Figure 5 shows the log10 transformed dNSAFs distribution of the 884 potato
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mitochondrial proteins. Each bin corresponds to 0.25 order of magnitude difference in
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the protein abundance. In this way, the potato mitochondrial protein abundance spans
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three orders of magnitude ranging from -4.82 to -1.57 log10 values (Table S3) giving a
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dynamic range of about 1800 fold as calculated by the normalized spectral count
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(dNSAF). This is likely a low estimate of dynamic range as the most abundant proteins
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may be underestimated. The least abundant protein detected in the potato proteome was
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a ribonuclease (GI 255538392) and the most abundant protein was a phosphate carrier
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protein (GI 255543593). Considering the previous mitochondrial study in Arabidopsis
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(Heazlewood et al., 2004), the current study significantly increased coverage of both
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low- and high-abundance proteins. For example, 53 predicted mitochondrial ribosomal
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proteins were confidently detected in at least two replicates in the current study with an
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average abundance of -2.68 [log10(dNSAF)] against only three ribosomal proteins
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detected in the previous Arabidopsis study (Heazlewood et al., 2004). Also, 52 proteins
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involved in the uptake of a variety of metabolites (ADP/ATP, dicarboxylate,
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fumarate/succinate, phosphate, malate) including membrane carriers, transporters, and
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porins were identified in the potato mitochondrial proteome with an average abundance
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of -3.31, while all previous Arabidopsis studies detected 19 proteins related to the same
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functional class. Proteins with well-known roles in primary mitochondrial metabolism
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including proteins involved in oxidative phosphorylation via the respiratory complexes
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and proteins from the TCA cycle were detected with an average abundance of -2.82
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comprising around 28% of total protein abundance. We also quantified low abundance
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proteins like those involved in RNA processing. For example, we quantified 62
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pentatricopeptide repeat (PPR) proteins, which have almost two orders of magnitude
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difference in expression levels (from -4.67 to -2.69 [log10(dNSAF)]) (Table S3).
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An overview of the number and abundance of the identified proteins belonging to major
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mitochondrial metabolic pathways are shown as heat maps in Figures 6 and 7. Figure 6
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shows the whole mitochondrion with processes placed in the known or presumptive
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mitochondrial subcompartment – OMM, intermembrane space (IMS), IMM, matrix –
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while Figure 7 shows the respiratory complexes and a few associated enzymes located
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in the IMM. Spectral counting should not be used to determine the stoichiometry of
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subunits in a complex, particularly membrane complexes. Having said that, the apparent
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wide variation in the amounts of the subunits, e.g. of Complex V, is mainly due to the
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presence of low-abundance paralogs (Table S3). If we compare the relative abundances
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of all the F1-ATPase subunits identified, they range between -3.72 and -1.96
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[log10(dNSAF)] or almost 100-fold (Table S3). However, if we only compare the most
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abundant paralogs of each of the five subunits identified, the range is much more
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narrow, between -2.91 and -1.96 [log10(dNSAF)], which is about 10-fold or about the
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same range as the expected 3:3:1:1:1 stoichiometry of the α, β, γ, δ, ε subunits (von
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Ballmoos et al. 2009).
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Prediction programs for mitochondrial localization recognize 63% of all identified
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proteins
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There are a large number of programs available to predict subcellular localization based
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on the N-terminal region of proteins that may contain intrinsic targeting peptides
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(Scheneider and Fechner, 2004). The protein sequences of the identified mitochondrial
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proteome were analyzed by five different prediction programs: TargetP (Emanuelsson et
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al., 2000), Predotar version 1.03 (Small et al. 2004), MitoProtII (Claros and Vincens,
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1996), iPSORT (Bannai et al., 2002) and WoLF PSORT (Horton et al., 2007). Each
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program employs different methods for subcellular prediction and usually produces
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relatively large non-overlapping sets of results (Heazlewood et al., 2004). Each
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prediction tool returned a variable percentage of mitochondrial positives ranging from
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approximately 20% (WoLF PSORT) to 52% (MitoProtII) of the potato proteome (Table
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S4). We also performed a relational evaluation of the overlapping positive predictions
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by different combinations of the prediction tools. When we considered multiple
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prediction tools together, the number of positive mitochondrial predictions decreased
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(Table S4), indicating a much smaller agreement between programs. The combination
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of MitoProt II and iPSORT provided the best pair for prediction resulting in 37%
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positive potato mitochondrial predicted proteins (Table S4).
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In all, 671 out of 1060 proteins (63% of the total protein set) were predicted to be
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mitochondrial by at least one program, while 48% were predicted by two or more
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programs (Fig. 8). From the list of 389 proteins not predicted to be mitochondrial by
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any program, 44 proteins were confirmed OMM or IMM proteins related to the protein
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import system and uptake of metabolites (carriers, transporters, channels, and
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translocases) which are characterized by the absence of N-terminal targeting
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presequences (Millar and Heazlewood, 2003; Huang et al., 2010). In addition, 31 well-
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known mitochondrial proteins involved in the TCA cycle and the respiratory complexes,
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as well as 11 proteins encoded by mitochondrial genes, were also negative for
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mitochondrial localization prediction. These results show the limitations in assigning
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mitochondrial localization based solely on bioinformatic tools. The choice of prediction
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program can benefit different classes of proteins and the absence of targeting
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presequences, due to genuine lack of those N-terminal targeting signaling sequences
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(e.g. OMM or IMM proteins or mitochondrially encoded proteins), or due to incorrect
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annotation of the N-terminus of the sequences, can result in erroneous interpretation.
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In Figure 8, the subcellular prediction information is superimposed on the relative
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protein abundance. The data reveal that prediction results are comparable irrespective of
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protein abundance, suggesting that low abundance proteins must also be considered as
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authentic mitochondrial proteins.
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Proteins with uncleaved N-terminal sequences are sometimes further processed
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Semi-tryptic database querying resulted in 37 matches to the protein N-terminus
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indicating that these proteins do not contain a cleavable targeting peptide. Some of these
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peptides had the terminal Met removed while others did not. The removal of the Met
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can be related to the length of the side chain of the second amino acid in the sequence
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(Gliglione and Meinnel, 2001). If the second amino acid is Ala, Gly, Pro, Ser or Thr, the
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initiating Met is removed by a Met aminopeptidase. On the other hand, if the side-chain
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is large, in case of Arg, Asn, Asp, Glu, Ile, Leu or Lys, the Met is retained. Only Val
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appears to have an intermediate-side-chain specificity for Met cleavage, showing the
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initiating Met cleaved or uncleaved. As shown in Table S5 our results confirm this
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specificity. The Met was removed when the second side chain was Lys, Ala, Pro, Ser,
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Thr or Val and not removed when it was Lys, Asn or Asp or Gln. Surprisingly, when the
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second residue was Gly, some sequences had Met removed while others did not.
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Many of the proteins identified with an uncleaved N-terminus were OMM proteins,
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including porins, or components of the respiratory complexes present in the IMM, such
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as NADH dehydrogenase, bc1 complex, and ATPase. Additionally, two mitochondrially
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encoded proteins were identified with intact N-termini, a ribosomal protein
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(STmpRH_41) and an NADH dehydrogenase subunit (STmpRH_8).
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The majority of proteins in the potato mitochondrial proteome are post-
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translationally modified
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The potato mitochondrial proteome was extensively searched for different types of post-
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translational modifications (PTMs) ( Tables S6 and S7). We identified 3,066 PTM sites
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in a total of 556 proteins which means that 52% of the entire potato mitochondrial
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proteome determined here exist in modified versions. Individual proteins contained up
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to eight types of PTMs and up to 50 different modification sites. The proteins with most
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modifications - aconitase, 2-oxoglutarate dehydrogenase, succinate dehydrogenase and
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glycine dehydrogenase - are all part of the TCA cycle or associated with it (Table S7).
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Mitochondria are not only the sites of oxygen consumption but also one of the sources
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of cellular reactive oxygen species (ROS) (Møller 2001). We therefore specifically
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focused our analyses on protein oxidation in the potato mitochondrial proteome.
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Oxidative modifications of Arg, Pro, Lys, Met, Thr, and Trp were detected. A total of
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505 proteins contained at least one type of oxidative modification giving a total of 2,471
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sites of modification. As shown in Figure 9, Met sulfoxidation followed by Pro
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oxidation (either hydroxylation or carbonylation) and Lys hydroxylation are the most
374
abundant types of PTM in the proteome. Most of the modified proteins participate in the
375
TCA cycle, respiratory complexes, protein import assembly, and protein synthesis
376
(ribosomal proteins) (Table S7). The high frequency of Met sulfoxidation is in
377
accordance with several reports that found sulfur amino acids to be more sensitive to
378
oxidation by ROS than other amino acids (Stadtman et al., 2005).
379
Carbonylated proteins include both high and low abundance proteins spanning a variety
380
of metabolic pathways (energy, metabolism, protein and DNA synthesis, protein fate
381
and transport). Among the highly abundant carbonylated proteins (abundance from -
382
2.99 to -1.56 log10[dNSAF]), up to 50 oxidation sites were mapped to a single protein,
383
glycine dehydrogenase (Table S7). Other examples were proteins involved in the TCA
384
cycle (aconitase, 2-oxoglutarate dehydrogenase, succinate dehydrogenase, citrate
385
synthase, isocitrate dehydrogenase), respiratory complexes (ATPase, cytochrome c1,
386
cytochrome c oxidase), chaperonins, carrier proteins (phosphate, oxoglutarate,
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387
ADP/ATP), pyruvate dehydrogenase complex (E1 α and β subunits) and redox
388
metabolism (peroxiredoxin, Mn superoxide dismutase, monodehydroascorbate
389
reductase). Many low-abundance proteins (-4.65 to -3.0 log10[dNSAF]) also showed a
390
large number of oxidation sites, up to 25 sites. Proteins with more than ten oxidation
391
sites include FtsH proteases, monoxygenase, ATPase and branched-chain α-keto acid
392
dehydrogenase (Table S7).
393
We also detected Asn deamidation (188 proteins), Lys acetylation (31 proteins) and
394
methylation (63 proteins), and Ser/Thr phosphorylation (19 proteins). These proteins
395
showed a wide range of relative abundance (-4.56 to -1.56 log10[dNSAF]) and
396
comprised components of TCA cycle and associated enzymes, chaperone system,
397
respiratory complexes, protein import system, RNA processing and carriers proteins.
398
399
DISCUSSION
400
The discord between the number of predicted and reported plant mitochondrial proteins
401
is strikingly high – probably more so than for any other organelle. Despite this
402
discordance, no shotgun proteomic study of plant mitochondria has been published
403
since 2005. Many advances in proteomics have occurred during the intervening time,
404
including the development of best practices for unbiased protein prefractionation,
405
sensitive and higher-accuracy mass spectrometers, as well as various refinements in
406
mass spectral data querying. In this study, we leverage developments on each of these
407
fronts to offer a quantitative, unbiased, and comprehensive proteome resource for the
408
plant mitochondrial community, including about 500 new experimentally identified
409
plant mitochondrial proteins. We estimate that the coverage of the mitochondrial
410
proteome is high (possibly as high as 85%) (see Supplementary Discussion). Since the
411
theoretical size of the plant mitochondrial proteome is about twice the size of the the
412
potato tuber proteome, it is likely that the specialized and physiological condition of the
413
tissue and its mitochondria placed a limit on the number of proteins identified. Thus, we
414
can discuss not only proteins found, but also comment with some confidence on
415
proteins or protein groups underrepresented. This will be a recurrent theme in the
416
following discussion as well as in Supplementary Discussion.
417
Prediction programs
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418
A comparison of five different subcellular localization prediction programs, TargetP,
419
Predotar, MitoProtII, iPSORT and WoLF PSORT), revealed a variable number of
420
mitochondrial predictions, ranging from 20 to 52% of the entire experimentally
421
determined mitochondrial proteome (Fig. 8, Table S4). These values are low compared
422
to the previous values of 70 to 90% reported in the original articles for these programs
423
(Emanuelsson et al., 2000; Small et al. 2004, Claros and Vincens, 1996). This could be
424
the result of bias in the protein training sets used in developing the programs. Also, we
425
found that the combination of multiple prediction programs showed low numbers of
426
overlapping positive predictions, indicating the divergence of the prediction methods
427
employed by each tool. Overall, 63% of the potato mitochondrial proteome was
428
predicted to be mitochondrially localized by at least one of the five prediction tools
429
(Figure 8, Table S4). However, authentic mitochondrial proteins involved in the TCA
430
cycle and the respiratory complexes as well as proteins characterized by the absence of
431
targeting presequences (OMM or IMM and mitochondrially encoded proteins) were
432
erroneously predicted to be non-mitochondrial. The number of such false negatives was
433
only partially offset by the use of different algorithms, indicating that the results
434
returned by such tools should be inspected carefully. False positives appear to be less of
435
a problem with these prediction programs. Nevertheless, even if a protein is not
436
predicted to be mitochondrial, one should not assume it is a contaminant as the accuracy
437
of these programs is both variable and limited. As most of these programs are decades
438
old, the development of new improved programs, based on more comprehensive
439
genomic sequences and proteomics data, is required to overcome the lack of accuracy
440
observed.
441
Basic metabolic processes
442
The coverage of the well-known mitochondrial functions - TCA cycle, respiratory
443
chain, transmembrane transporters, and turnover of amino acid, proteins, lipids and fatty
444
acids - was high as shown in Figures 6 and 7 and listed in Table S1. A detailed
445
discussion of the potato tuber proteins identified for each group as well as for glycolysis
446
and signaling is found in Supplementary Discussion. In the following we will discuss a
447
number of interesting protein groups linked to other important mitochondrial functions.
448
Biosynthesis of coenzymes and pyrimidines
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449
Vitamins are coenzymes, which humans are unable to synthesize but acquire through
450
their food, mostly from plants. Mitochondria are involved in the biosynthesis of several
451
of the coenzymes in plant cells and we have found a number of key enzymes in these
452
processes, some of which have previously only been predicted.
453
Although NAD+ is the main pyridine coenzyme in mitochondria, a number of enzymes
454
use NADP+ (Møller and Rasmusson 1998). NAD+ uptake into plant mitochondria was
455
discovered in the 1980’s using POM (Neuburger et al. 1985), but NADP+ uptake has
456
also been inferred at least for pea leaf mitochondria (Bykova and Møller 2001). We here
457
identify an NAD+ carrier as well as an NADH kinase (Table S1). Three NAD(H)
458
kinases are found in Arabidopsis and one of them, NDK3, which appears to prefer
459
NADH as substrate over NAD+, has been reported to be localized to peroxisomes in
460
higher plants while its analogue in yeast is mitochondrial (Waller et al. 2010). The POM
461
NADH kinase is predicted to be mitochondrial by three prediction programs and is of
462
relatively low abundance (-3.87 [log10(dNSAF)]) (Tables S1 and S3, Fig. 6). Thus, in
463
order to generate the NADP+/NADPH pool inside the POM, it appears that NAD+ is
464
taken up, reduced by one of the many matrix dehydrogenases, e.g. in the TCA cycle,
465
and then phosphorylated to give NADPH. This does not imply that NADPH/NADP+
466
ratios in the mitochondria are determined by this pathway, rather that generation of the
467
overall NADP+/NADPH pool size would be governed by the activities of the NAD+
468
transporter and the NADH kinase.
469
L-Galactono-1,4-lactone dehydrogenase, the last enzyme in one of the ascorbate
470
biosynthetic pathways, which donates electrons to Complex IV on the outer surface of
471
the IMM (Bartoli et al. 2000), but is physically associated with Complex I (Pineau et al.
472
2008), was found at above median quantities. Five different enzymes using ascorbic
473
acid mainly for ROS detoxification were also found (see later). Clearly ascorbate must
474
be imported in some form, but the mitochondrial carrier protein family does not appear
475
to include a carrier for ascorbate or dehydroascorbate (Taylor et al. 2010, Palmieri et al.
476
2011) and no such carrier was found in our study.
477
Tetrahydrofolate (vitamin B9) and its derivatives catalyze the addition or removal of
478
C1-units, e.g., in the glycine decarboxylate reaction in the mitochondrial matrix where a
479
methyl group is transferred from one glycine molecule to another to form serine. Folates
480
consist of a pterin moiety, a p-aminobenzoate moiety and a (poly)glutamate tail and the
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481
three parts are assembled in the mitochondria (Blancquaert et al. 2010). In the POM, we
482
have found three enzymes involved in folate biosynthesis, dihydropterin
483
pyrophosphokinase-dihydropteroate synthase, a molybdopterin biosynthesis protein and
484
polypolyglutamate synthase, four enzymes involved in the interconversion and transfer
485
of different C1-units, 5-formyltetrahydrofolate cycloligase, formyltetrahydrofolate
486
deformylase-like, methenyltetrahydrofolate synthase domain-containing protein-like, 5-
487
methyltetrahydropteroyltriglutamate-homocysteine methyltransferase-like and finally a
488
folate carrier responsible for exporting folate to make the coenzyme available to the rest
489
of the cell.
490
Biotin (vitamin B8) is a coenzyme involved in carboxylations and the last steps in its
491
biosynthesis are mitochondrial (Alban 2011). We found the S-adenosylmethionine
492
carrier responsible for the import of one of the precursors, but not predicted to be
493
mitochondrial by any of the prediction programs. We also detected the adrenodoxin
494
reductase involved in biotin biosynthesis and predicted to be mitochondrial by three
495
programs (Table S1). The adrenodoxin reductase uses adrenodoxin to reduce S-S
496
bridges and we find five ferredoxin analogs, which may well be adrenodoxin. Thus, a
497
significant part of the biotin biosynthesis pathway is expressed in POM.
498
Pyrimidine biosynthesis mostly takes place in the cytosol and the nucleus in mammalian
499
cells, but the fourth reaction in the pathway, catalyzed by dihydroorotate
500
dehydrogenase, takes place on the outer IMM surface (Löffler et al. 2005). Heazlewood
501
et al. (2004) found this enzyme in Arabidopsis mitochondria and in POM it is present at
502
higher than median relative abundance (Table S3) confirming that it is mitochondrial
503
and probably donates its electrons to ubiquinone as in mammalian mitochondria (Fig.
504
7).
505
The identification in this one investigation of so many enzymes involved in coenzyme
506
biosynthesis in the mitochondrion is a good demonstration of the power of applying
507
high resolution and highly sensitive mass spectrometry-based proteomic tools to
508
important questions in plant biochemistry.
509
DNA and RNA
510
We identified DNA polymerases, RNA polymerases, RNA helicase, histones, an
511
histone-modifying enzyme, a topoisomerase, transcription factors and a transcription
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512
termination factor, most at low abundances (Table S1 and S3) and representing almost
513
all of the components required for DNA replication and transcription. Three of the five
514
histones identified are of a type recently reported to be present in plant mitochondria
515
(Zanin et al. 2010).
516
Pentatricopeptide repeat (PPR) proteins
517
PPR proteins are one of the most prolific protein families in plants while it is virtually
518
absent in animals (Small and Peeters 2000). The majority of the 450 PPR proteins in
519
Arabidopsis are predicted to be mitochondrial where about half are thought to be
520
involved in RNA editing and the remainder in other types of RNA processing (Fujii and
521
Small 2011). In Arabidopsis mitochondria, Heazlewood et al. (2004) found 10 PPR
522
proteins while in potato tuber mitochondria we found 71. From the total of 71 PPR
523
proteins, we relatively quantified (dNSAF) 62 PPR proteins found in at least two
524
replicates. The abundance of these PPR proteins varied by almost two orders of
525
magnitude based on dNSAF values. Only 7 PPR proteins can be considered to be
526
present at high relative abundance (-2.9 to -2.6 [log10(dNSAF)]), whereas 23 can be
527
considered medium relative abundance (-3.9 to -3.0 [log10(dNSAF)]) and 32 low
528
relative abundance (-4.67 to -4.0 [log10(dNSAF)]).
529
Since 450 PPR proteins are present in Arabidopsis (Fujii and Small 2011) and since the
530
potato tuber genome contains many more genes than the Arabidopsis genome, it is very
531
likely that at least 300 PPR proteins are targeted to the mitochondria in some potato
532
tissue under some condition. In spite of the fact that all proteins encoded in mtDNA are
533
subunits in the constitutively expressed respiratory complexes or appear to be essential
534
housekeeping proteins, the finding of “only” 71 PPR proteins indicates that many RNA-
535
related processes are inactive in POM. In mitochondria from perennial ryegrass
536
reproductive tissues, a transcript profile showed that the expression of several
537
mitochondrially expressed ribosomal proteins was very low (Islam et al. 2013). This
538
may mean that their expression, and the associated RNA-related processes including
539
PPR proteins, is dependent on the tissue and/or environmental conditions.
540
Turnover of Reactive Oxygen Species
541
The mitochondrion is one of the major sites of ROS production in the cell (Maxwell et
542
al. 1999, Møller 2001, Foyer and Noctor 2003) and it therefore also contains a number
19
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543
of enzymes or enzyme systems capable of removing ROS. We found three of the four
544
enzymes in the ascorbate-glutathione pathway, glutathione reductase, ascorbate
545
peroxidase, and monodehydroascorbate reductase (dehydroascorbate reductase was not
546
detected) (Jimenez et al. 1998, Chew et al. 2003) with very similar relative abundances
547
(-3.9 to -3.6 [log10(dNSAF)]), except for one monodehydroascorbate reductase (GI
548
350536875) which presented higher relative abundance (-2.8 log10(dNSAF)]) (Fig. 5).
549
Also, we have identified both the classical mitochondrial Mn-SOD as well as a Cu,Zn-
550
SOD, which at least in yeast is found in the mitochondrial intermembrane space
551
(O’Brien et al. 2004). Mn- and Cu,Zn SODs showed high (-1.7 [log10(dNSAF)]) and
552
medium (-3.5 [log10(dNSAF)]) relative abundances, respectively.
553
We found thioredoxin (-3.1 [log10(dNSAF)]) and thioredoxin reductase, phospholipid
554
hydroperoxide glutathione peroxidase (-3.2 log10[dNSAF]) and peroxiredoxin (-2.6
555
log10[dNSAF])) – all in medium relative abundance. In other words, all the four
556
predicted NADPH-consuming enzyme systems involved in removing H2O2 or H2O2-
557
induced peroxidation products (Møller 2007) are expressed in POM, which appear well
558
armed to deal with oxidative stress.
559
Finally, we detected two catalases (one at low level – 4.4 and one at medium level (-2.6
560
[log10(dNSAF)]). Although POM only contain a maximum of 0.2% peroxisomes on a
561
protein basis, the catalases could be contaminants, since catalase makes up
562
approximately 50% of all the peroxisomal protein in potato tubers (Struglics et al.
563
1993). However, catalase has been reported to be present inside rat heart mitochondria
564
(Radi et al. 1991) so the parsimonious explanation of catalase inside plant mitochondria
565
cannot be excluded.
566
Metal ion turnover
567
Mitochondria contain many proteins, particularly redox active enzymes, which bind
568
metal ions often at their catalytic site. As mitochondria are also involved in the
569
biosynthesis of FeS centers and hemes, the concentration of metal ion is high in
570
mitochondria (Tan et al. 2010). However, because free metal ions can interact with
571
hydrogen peroxide and form the highly reactive and damaging hydroxyl radical, which
572
can lead to protein oxidation (see below), it is very important for the cell to keep metal
573
ions safely bound to proteins at all stages of metal ion uptake and metabolism (Kell
574
2009, 2010, Møller et al. 2011). We found a number of prominent metal-containing
20
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575
proteins including the FeS-proteins and the Cu/Fe-containing cytochromes in the ETC,
576
but also Mn- and Cu/Zn-SOD and TCA cycle enzymes like aconitase (Fe). In addition,
577
we found the Fe-storing ferritin (low abundance) and the complete pathway for the
578
biosynthesis of FeS clusters – frataxin, iron-sulfur cluster assembly proteins, iron-sulfur
579
cluster scaffold protein, iron-sulfur cluster co-chaperone protein, cysteine desulfurase
580
and ferredoxin (all medium abundance) (Ye and Rouault 2010).
581
A majority of potato mitochondrial proteins are post-translationally modified
582
More than half the proteins detected in the potato mitochondrial proteome were post-
583
translationally modified on at least one site. And about 100 proteins had more than ten
584
PTM sites. The most modified protein, glycine dehydrogenase, had as many as 50 PTM
585
events of six different kinds including 24 Met oxidations, 3 Asp deamidations and 12
586
Pro oxidations (Table S7). In most cases the spectral counts were very low for each
587
modified peptide identified. It is likely that the coverage of modified peptides were
588
underestimated as PTMs such as Lys or Arg oxidation causes missed trypsin cleavage
589
sites resulting in larger peptides that are more difficult to ionize and fragment.
590
The large number of PTM sites on some proteins could give rise to a very large number
591
of differentially modified versions of the protein unless the PTMs are introduced in an
592
ordered rather than a random manner (Thelen and Miernyk, 2012). A relatively
593
unexplored research area is the interaction between different PTMs. At least in one case
594
we know that such an interaction takes place – The oxidation of a Met residue to Met-
595
sulfoxide next to a potential phosphorylation site abolishes phosphorylation on that site
596
and this is likely a regulatory mechanism as this first step in Met oxidation is reversible
597
(Huang et al., 2010).
598
In Supplementary Discussion we analyze phosphorylated proteins, oxidized proteins
599
and proteins with acetylated and methylated lysines.
600
Conclusions
601
A comprehensive shotgun proteomic investigation of potato tuber mitochondria led to
602
the detection of 1060 proteins, which appear to represent more than 85% of the proteins
603
expressed in the POM. A significant disparity was observed between the proteins
604
actually identified in the proteome and those predicted to be targeted to the proteome by
605
the five most commonly used prediction algorithms. Less than 50% of the proteins
21
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606
determined to be present in the mitochondrial proteome were predicted to be thus
607
localized by at least two of these programs. Indeed, only 63% were thus predicted by at
608
least one program. This clearly demonstrates that current bioinformatics tools, while
609
often useful, can be very inadequate at predicting biological function and localization.
610
The broad coverage observed in the POM proteome allowed for investigation of
611
processes that are important for mitochondrial function, such as metabolism and
612
respiration and post-translational regulation of protein function. The ETC was well
613
covered in the POM proteome, with all complexes being well represented. In addition,
614
three different alternative NAD(P)H dehydrogenases were present at significant levels,
615
as were all members of the TCA cycle, except fumarase, suggesting that non-cyclic
616
modes of operation are likely to be functional. In addition, the biosynthesis of many
617
coenzymes was well represented in the proteome, supporting the role of the
618
mitochondrion in production of these important molecules. The general processes of
619
amino acid biosynthesis and lipid metabolism were well supported in the proteome. The
620
fact that a number of glycolytic enzymes were detected can be explained by their
621
association with the OMM. A number of other processes important to mitochondrial
622
function and replication were well supported, including transport, protein synthesis,
623
regulation of gene expression within the mitochondria, and metal ion regulation.
624
Finally, a large number of protein PTMs were found in the mitochondrial proteome,
625
with over 50% of proteins possessing such modifications and several proteins
626
possessing over a dozen modifications, such as phosphorylation and oxidative
627
modifications. The ease with which these modifications were found is a testament to the
628
power of the approach used. The fact that so many proteins contained at least one, and
629
many contained more than one PTM, suggests that this mode of functional regulation
630
plays an important role in the mitochondria. It further suggests that approaches beyond
631
transcriptional profiling will be not only desired, but required, if we are to be able to
632
better understand how mitochondria help plants respond to stress and changing
633
environments.
634
MATERIALS AND METHODS
635
Isolation of mitochondria
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636
Mitochondria were isolated from potato (Solanum tuberosum L., cv. Folva) tubers by
637
the method of Considine et al. (2003) with minor modifications. All procedures were
638
done at 4°C. Peeled tubers were homogenized using a juice extractor into an equal
639
volume of extraction medium (0.6 M mannitol, 20 mM MOPS-KOH (pH 7.3), 2 mM
640
EDTA, 25 mM cysteine and 0.3 % (w/v) BSA). The homogenate was left standing for 5
641
min allowing starch to sediment before it was filtered through 2 layers of cotton towel
642
and centrifuged at 3000 g for 5 min. The supernatant was centrifuged at 18000 g for 10
643
min to recover an organelle pellet. The pellet was resuspended in mannitol buffer (0.3
644
M mannitol, 10 mM MOPS-KOH (pH 7.3), 0.1 % (w/v) BSA) and layered on top of a
645
Percoll step gradient of 50 %, 28 % and 20% in mannitol buffer. After centrifugation at
646
40000 g for 30 min the pale mitochondrial band was recovered from the 28%/50%
647
Percoll interface. After washing, the mitochondria were further purified on a second
648
self-forming Percoll gradient of 28% Percoll in sucrose buffer (0.3 M sucrose, 10 mM
649
MOPS-KOH (pH 7.3), 0.1 % (w/v) BSA). The protein concentration was estimated by
650
measuring absorbance at 280 nm using a NanoDrop 1000 Spectrophotometer (Thermo
651
Scientific).
652
Gel electrophoresis
653
One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS–
654
PAGE, 20 × 20 cm) performed according to (Laemmli 1970) was employed as a pre-
655
fractionation step. Isolated mitochondria (250 µg) and size marker proteins (protein
656
ladder, Thermo Scientific # 26630) were dissolved in sample buffer (NuPAGE®),
657
placed at 65°C for 15 min and resolved by 12% SDS-PAGE. After protein migration,
658
proteins were stained with colloidal Coomassie Brilliant Blue. Then, each gel lane was
659
sliced in 40 segments and diced in approximately 1 mm cubes followed by gel
660
destaining and in gel digestion as described by Balbuena et al. (2011).
661
Liquid chromatography-mass spectrometry
662
A two column system was used. Both columns were in-house packed with C18 reverse
663
phase material (ReproSil, C18 AQ 3 μm (Dr. Maisch, Germany)) in fused silica. The
664
pre-column had an inner diameter of 100 μm and a length of 2 cm. The analytical
665
column had an inner diameter of 75 μm and a length of 15 cm. The flow of 250 nl/min
666
was delivered by an EASY nLC system (Thermo Fisher Scientific, Odense, Denmark)
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667
and the peptides were eluted using a 21 min gradient from 0% to 40% B buffer (A
668
buffer: 0.1% formic acid, B buffer: 0.1% formic acid/90% acetonitrile, all v/v). The
669
flow from the analytical column was coupled to an LTQ-Orbitrap XL mass
670
spectrometer (Thermo Fisher Scientific). The instrument was run in positive ion mode.
671
Full scan MS spectra (from m/z 300-1800) were acquired in the Orbitrap with resolution
672
r = 60,000 at m/z 400. The up to 5 most intense ions with charge states larger than +1
673
were sequentially isolated for fragmentation in the linear ion trap using collision-
674
induced dissociation. Former target ions selected for fragmentation were dynamically
675
excluded for 30 s. For accurate mass measurements the lock mass option was enabled in
676
MS mode and the polydimethylcyclosiloxane (PCM) ions generated in the electrospray
677
process (m/z = 445.120025) were used for internal recalibration in real time, resulting in
678
a mass accuracy of approximately 2 ppm.
679
Database searching and protein identification
680
Acquired MS/MS spectra were searched using different algorithms against the potato
681
protein database (http://www.potatogenome.net). To estimate the protein false discovery
682
rate (FDR), randomized sequences were combined with the forward database in a
683
concatenated format. MASCOT server 2.3.01, SEQUEST, MS-GFDB (Kim et al.,
684
2010) and ProLuCID (Xu et al., 2006) were used to interpret the data set using similar
685
parameters. MASCOT and SEQUEST were integrated within the Proteome Discoverer
686
1.3 software package (Thermo Scientific). Raw files (Thermo) were converted to ms2
687
and mzXML files prior to analysis by ProLuCID and MS-GFDB. All search engines
688
were configured to the following parameters: MW range between 200 and 2,000; 1,000
689
ppm of precursor ion tolerance; fragment tolerance 1.0 Da oxidation of methionine and
690
asparagine deamidation as variable modifications and carbamidomethylation of cysteine
691
as a static modification; two allowed missed trypsin cleavages. After searches, the
692
PSMs were filtered with a 5 ppm precursor ion tolerance, achieving a false discovery
693
rate (FDR) lower than 1% at the protein level for each biological replicate.
694
All the MS data from each gel slice was searched and merged for each replicate. The
695
results from different programs were combined in order to complement observations
696
that came from different processes of data mining, without compromising the accuracy.
697
To do this, a high stringency on precursors (5 ppm) was applied.
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698
Filtered data were uploaded to the Protein Herder module within the Compass package
699
(Wenger et al., 2011) for protein grouping. At this step, all sets of indistinguishable
700
proteins, which were identified by the same peptides, were combined into protein
701
groups.
702
Purity assessment of mitochondrial preparations
703
Total protein and mitochondrial protein-enriched fractions from potato tubers were
704
separated by SDS-PAGE. From each fraction, 20 μg of proteins was loaded on the gel.
705
Blotting was perfomed as previously described by Stevenson et al. (2009) to PVDF
706
membranes (GE Healthcare Ltd, UK). Antibodies directed against 14-3-3, enolase,
707
plastidic alpha carboxytransferase (α-CT) and mitochondrial pyruvate dehydrogenase
708
E1 component subunit alpha (PDE1-α) were used to assess mitochondrial protein
709
enrichment in the preparations. Immunodetection of proteins bound to PVDF
710
membranes was performed using the colorimetric alkaline phosphatase and peroxidase
711
substrate detection system (Sigma-Aldrich).
712
Subcellular Localization Study
713
Total RNA was isolated from potato tuber by using the method described by Kumar et
714
al., (2007). A cDNA library was then synthesized by M-MLV reverse transcriptase
715
reaction (Promega, WI, US) with random hexamer. Potato cDNA was PCR amplified
716
from cDNA library by a pair of gene-specific primers; BamHI and Not I restriction
717
enzyme cutting sites were added to forward and reverse primers, respectively (Table
718
S8). The PCR fragments were cloned into pGEM-T easy vector (Promega, WI, US), the
719
insertions were dropped off by BamHI and NotI double digestion, and the insertions
720
were cloned into pE6c entry vector, such that an EYFP fluorescence tag was fused in
721
frame with potato proteins at the C-terminus (Dubin et al., 2008). The cassette was then
722
moved into the binary vector pSITE-0B by Gateway LR clonase (Chakrabarty et al.,
723
2007).
724
For the co-localization assay, mitochondria marker (CD3-986) binary plasmid with CFP
725
fluorescence tag was ordered from the Arabidopsis Biological Resource Center (Nelson
726
et al., 2007). The binary constructs were transformed into Agrobacterium AGL1 strain.
727
The potato EYFP fusion construct was individually mixed with the CFP mitochondrial
728
marker and co-transformed into tobacco leaves. Transformation was performed by
25
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
729
following the procedure described by Sparkes et al. (2006). An Olympus IX70
730
microscope controlled by MetaMorph (Version 6.3 v6, Molecular Device) software was
731
used for image capture, and Chroma filter 49001 and 49003 were applied for the CFP
732
and EYFP channel, respectively.
733
Post-translational modifications
734
Multiple post-translational modifications (PTM) were searched against a concatenated
735
database containing forward and randomized protein sequences previously identified in
736
the general proteome searching approach. The searches were performed using
737
SEQUEST, MASCOT and MS-GFDB programs. For each round of PTM searching a
738
maximum of two different variable modifications (PTMs) were configured. Precursor
739
ion tolerance was set to 100 ppm and 2 missed tryptic cleavages were allowed.
740
Carbamidomethylation of cysteine was configured as a static modification and the
741
PTMs of interest were configured as variable modification. After searches, the data set
742
was filtered using 5 ppm as precursor ion tolerance, achieving FDR lower than 1%.
743
Only PTMs detected in at least 2 replicates were considered. The spectra assigned to
744
peptides with oxidation sites were searched against the database allowing all types of
745
oxidation sites at the same time to make sure that the oxidation sites were reproducible
746
compared to the first database search.
747
Semi-tryptic database searching
748
All MS data was reanalyzed using MS-GFDB program against the identified protein
749
sequences database allowing semi-tryptic peptides and 100 ppm of precursor ion
750
tolerance. Carbamidomethylation of cysteine was configured as a static modification
751
and methionine oxidation as variable modification. The PSMs obtained were filtered out
752
using 5 ppm as precursor ion tolerance, resulting in a FDR lower than 1%.
753
Prediction of Subcellular localization
754
Predictions of subcellular localization for identified proteins were taken using the full-
755
length protein sequences and five programs namely TargetP version 1.1
756
(http://www.cbs.dtu.dk/services/TargetP/, Emanuelsson et al., 2000), Predotar version
757
1.03 (http://urgi.versailles.inra.fr/predotar/predotar.html, Small et al. 2004), MitoProtII
758
(http://ihg.gsf.de/ihg/mitoprot.html, Claros and Vincens, 1996), iPSORT
26
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
759
(http://ipsort.hgc.jp/, Bannai et al., 2002) and WoLF PSORT (http://wolfpsort.org/,
760
Horton et al., 2007).
761
Molecular weight and isoelectric point
762
Molecular weight and isoelectric point of proteins were obtained using Compute pI/Mw
763
web server (http://web.expasy.org/compute_pi/, Bjellqvist et al. 1994, Gasteiger et al.
764
2005). The grand average hydropathy (GRAVY) score was computed based on Kyte-
765
Doolittle hydropathy scale (Kyte and Doolittle 1982). Bioinformatics computations
766
were performed using Python ver. 2.7. Arabidopsis mitochondrial proteome list is based
767
on Supplementary Table 1 in Heazlewood et al. (2004).
768
Protein abundance calculation
769
Total number of PSM (peptide spectrum matched) for each protein identified was
770
extracted from Protein Herder module of Compass software (Wenger et al., 2011) for
771
each biological replicate. Only proteins identified in at least two replicates were
772
considered for quantification based on the distributed normalized spectral abundance
773
factor approach (dNSAF, Zhang et al., 2010). Spectral counts (SpC) for each protein or
774
group of proteins were used to estimate protein abundance.
775
For protein abundance estimation, the PSMs produced by each program were unified by
776
an in-house developed script. Only different scans for a given protein detected by
777
multiple programs were considered for calculation of total spectral counts, which means
778
that repeated scans were considered only once in the calculation of total spectral counts.
779
Therefore, the total spectra counts of each protein was calculated by the sum of PSMs
780
(non-redundant scans) produced by different algorithms.
781
The dNSAF considers the spectral counts of shared peptides and distributes them based
782
on a distribution factor (d):
783
dk =
784
where k denotes a protein identity and n is the total number of proteins, while uSpC
785
represents the sum of spectra matched to peptides mapping uniquely to the respective
uSpCk
∑uSpCn
27
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
786
protein. So, for a given protein k, d is equal to the total uSpC mapped to this protein
787
divided by the total number of uSpC from all n proteins with protein k shares peptide(s).
788
Then, the dNSAF for a given protein, k, is calculated as follows:
789
dNSAFk =
[(uSpC + d × sSpC ) ÷ L]k
∑ [(uSpC + d × sSpC) ÷ L]n
n
790
where sSpC corresponds to the total number of spectra from peptides shared among
791
related proteins and L represents the length of the protein. In this way, the shared
792
spectral counts (sSpC) are distributed amongst the proteins, avoiding that the same
793
spectrum will be counted multiple times.
794
Annotation and functional classification
795
Protein annotation and classification were performed based on matching the protein
796
sequences against the non-redundant NCBI database and Gene Ontology functional
797
classification using the Blast2GO tool (Conesa et al., 2005). Protein sequences were
798
aligned using the BLASTP algorithm against nr NCBI database using the following
799
parameters: report a maximum of 5 blast hits, 0.1 for the expected value and minimum
800
high scoring segment pairs (HSPs) length equal to 33. Mapping and annotation steps
801
were performed using Blast2GO default values. GOslim annotation was performed
802
using the plant slim. GO distribution graphs related to biological process and molecular
803
function were generated and analyzed at the fourth level of depth. Also, each protein
804
was assigned to general functional categories according to Heazlewood et al. (2004).
805
806
Acknowledgements
807
JFH is grateful to Drs. Katherine Beard and Lee J. Sweetlove for a study visit to their
808
laboratory at University of Oxford.
809
Authorship
810
JFH, JJT and IMM designed the research, JFH, MC and JJT performed the experiments,
811
FS, JFH, JJT, RSPR, and IMM contributed new computational tools and analyzed the
812
data, and FS, JFH, ARW, ONJ, DRG, JJT and IMM wrote the paper.
28
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
813
814
Supplemental Data
815
Supplementary Discussion
816
Figure S1: SDS-PAGE and corresponding immunoblots of enriched mitochondrial
817
protein fraction and total protein extract from potato tubers.. A, Commassie blue-stained
818
SDS-PAGE gel loaded with 20 µg of protein per lane. Positions of protein standards are
819
shown on the left (in kilodaltons). B, protein blots of mitochondrial enriched fraction
820
and total protein extract (fractions in A). The molecular masses of the protein bands are
821
shown on the left (in kilodaltons)
822
Figure S2: Subcellular localization study on a selected subset of proteins identified.
823
See Figure 2 and Table S2 for details.
824
Figure S3:. GO term distribution related to biological process and molecular function of
825
the proteins identified in the potato tuber mitochondria.
826
Table S1: List of 1060 protein groups identified by GeLC-MS/MS in isolated
827
mitochondria from Solanum tuberosum tubers.
828
Table S2: List of selected candidate proteins for validation by subcellular localization in
829
tobacco leaf.
830
Table S3. Label-free quantification based on spectral counting (884 proteins).
831
Table S4. Mitochondrial localization prediction results (for potato and Arabidopsis
832
mitochondrial proteomes) with different combinations of prediction programs.
833
Table S5: Proteins with non-cleaveable N-terminal presequences identified by LC-
834
MS/MS.
835
Table S6: Monoisotopic mass changes used to identify the post-translational
836
modifications.
837
Table S7: Posttranslational modifications detected in the mitochondrial proteome of
838
Solanum tuberosum.
29
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
839
Table S8: List of forward and reverse primer sequences used in this study for gene
840
cloning.
841
Repository data
842
Raw files as well peptide and protein identification files exported for each search engine
843
(SEQUEST, MASCOT MSGF-DB and ProLuCID) have been deposited in a public
844
repository - the ProteomeXchange Consortium
845
(http://proteomecentral.proteomexchange.org) via the PRIDE partner repository
846
(Vizcaino et al. 2013) with the dataset identifier PXD000149 available at
847
http://www.ebi.ac.uk/pride/. Phosphopeptides and respective spectra data have been
848
deposited at P3DB (Plant Protein Phosphorylation Database) available at
849
http://p3db.org/PotatoMitochondriaPhosData.php.
850
851
852
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1215
46
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
1216
FIGURE LEGENDS
1217
Figure 1: Workflow chart for the isolation of mitochondria from potato tubers
1218
followed by protein separation, identification and spectral counting-based
1219
quantification. First, mitochondria were isolated from potato tubers using Percoll
1220
gradients. Then, mitochondrial proteins were extracted and fractionated by SDS-
1221
PAGE. After gel segmentation, in gel trypsin digestion was performed and peptides
1222
were injected into an LC system coupled to a mass spectrometer. Database searching
1223
was carried out using four different algorithms (MSGF-DB, ProLuCID, Mascot,
1224
Sequest) and the potato genome database (http://www.potatogenome.net). The number
1225
of assigned MS/MS spectra for each identified protein was determined and the
1226
distributed normalized spectral abundance factor was calculated for protein abundance
1227
estimation.
1228
Figure 2: The number of peptides (A) and proteins (B) identified using different
1229
database search engines with a <1% protein FDR identification. The total number of
1230
non-redundant peptides and proteins were 7346 and 1060, respectively.
1231
Figure 3: Subcellular localization study on a selected subset of proteins identified in
1232
this study. Twenty proteins that were identified in purified potato tuber mitochondria
1233
(Table S1) were in-frame fused with EYFP fluorescence tag, then transiently co-
1234
expressed with the mitochondrial marker CD3-986 in tobacco epidermal cells. The
1235
left lane shows the EYFP fluorescence of six out of the 19 constructs where
1236
expression was detected. The center lane shows the fluorescence of the mitochondrial
1237
marker. The right lane shows an overlay between the two types of fluorescence and
1238
green colour indicates coincidence of the two probes, i.e., mitochondrial localization.
1239
The remaining 13constructs are shown in Figure S2. Size bar, 12.5 μm.
1240
Figure 4: Properties of the potato mitochondrial proteome (n = 1060) as compared to the
1241
Arabidopsis mitochondrial proteome (n = 416; [Heazlewood et al. 2004]). (A)
1242
Distribution of molecular mass (kDa), (B) isoelectric point and (C) Grand Average
1243
Hydropathy (GRAVY) score. A disproportionately large number of low-molecular-
1244
weight proteins with negative GRAVY scores were identified in the potato
1245
mitochondrial proteome.
47
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
1246
Figure 5: Abundance distribution of the potato mitochondrial proteome. Mitochondrial
1247
localization prediction results are superimposed on the protein abundance (spectral
1248
counting) [log10(dNSAF)]. Plus signs (+) indicate mitochondrial localization by as many
1249
as five prediction programs and minus sign (-) indicates localization by none. Each
1250
category on the x-axis corresponds to 0.25 order of magnitude difference in the
1251
abundance of proteins. The difference between the most abundant and the least
1252
abundant proteins was 1778 fold. The abundance calculation for all proteins is found in
1253
Table S3.
1254
Figure 6: Schematic representation showing proteins that were identified by GeLC-MS-
1255
MS in the mitochondrial preparation of potato tubers. Squares represent protein
1256
identification and abundance based on spectral counting. Each color represents a range
1257
of Log10 transformed dNSAF (bottom left). Numbers in parentheses represent number
1258
of proteins found within the same range of abundance. Respiratory chain complexes
1259
(OXPHOS) are shown in detail in Figure 6. Black text: metabolic intermediates; grey
1260
text: enzymes or proteins. Abbreviations: ABC, ABC transporter; AC, aconitase;
1261
ACOT, acyl-coenzyme A thioesterase 9; ACS, Long chain acyl-CoA synthetase 9APX,
1262
ascorbate peroxidase; AOX, alternative oxidase; ASC; ascorbate; AlaAT, alanine
1263
aminotransferase; AspAT, aspartate aminotransferase; BCKDH, branched chain alpha-
1264
keto acid dehydrogenase; CAC, carnitine/acylcarnitine carrier protein; CI-CV,
1265
respiratory chain complexes (see Figure 7); CoA DHase, isovaleryl-CoA
1266
dehydrogenase; CS, citrate synthase; DiT1, oxoglutarate malate transporter; DHA,
1267
dehydroascorbate; DHAR, dehydroascorbate reductase; DIC, Mitochondrial
1268
dicarboxylate carrier protein; D-LDH, D-lactate dehydrogenase; E1α, pyruvate
1269
dehydrogenase subunit α; E1β, pyruvate dehydrogenase subunit β; E2,
1270
dihydrolipoamide S-acetyltransferase; E3, Dihydrolipoamide dehydrogenase; ECR,
1271
trans-2-enoyl-CoA reductase; EH, Enoyl-CoA hydratase; ER, enoyl-acyl-carrier-protein
1272
reductase; ETF, electron transfer flavoprotein; FCL, 5-formyltetrahydrofolate
1273
cycloligase; FDF, formyltetrahydrofolate deformylase-like; FUM, fumarase; GABA, γ-
1274
aminobutyric acid; GABA-T, GABA transaminase; GDC, glycine decarboxylase
1275
complex; GDH, glutamate dehydrogenase; Gly-II, hydroxyacylglutathione hydrolase 3;
1276
Gly-I, lactoylglutathione lyase; GPX, glutathione peroxidase; GR, glutathione
1277
reductase; GRX, glutaredoxin; GSH; reduced glutathione; GSSG; oxidized glutathione;
1278
GST, glutathione-s-transferase; HAD, hydroxyacyl-thioester dehydratase; HMG lyase,
48
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
1279
hydroxymethylglutaryl-CoA lyase; ICDH, isocitrate dehydrogenase; IscA, iron sulfur
1280
cluster assembly protein; MCAT, acyl-carrier-protein S-malonyltransferase; MCC, 3-
1281
methylcrotonyl-CoA carboxylase; MCD, malonyl CoA decarboxylase; MCP,
1282
mitochondrial carrier proteins; MG hydratase, methylglutaconyl-CoA hydratase; MDH,
1283
malate dehydrogenase; MDHA, monodehydroascorbate; MDHAR,
1284
monodehydroascorbate reductase; MFT, mitochondrial folate transporter/carrier-like;
1285
MnSOD, superoxide dismutase; MPP, mitochondrial processing peptidase; NAD-ME,
1286
NAD-dependent malic enzyme; NDT, nicotinamide adenine dinucleotide transporter; 2-
1287
OGDH, 2-oxoglutarate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PGP,
1288
phospholipid hydroperoxide glutathione peroxidase; PHB, prohibitin; Pic, phosphate
1289
carrier protein; PRDX, peroxiredoxin; PSP, presequence protease; SAM, S-
1290
adenosylmethionine; SAM-T, S-adenosylmethionine-dependent methyltransferase;
1291
SAMC, S-adenosylmethionine carrier protein; SDH, succinate dehydrogenase; SCaMC,
1292
calcium-binding mitochondrial carrier protein; SFC, Succinate/fumarate mitochondrial
1293
transporter; SHMT, serine hydroxymethyltransferase; SSA, succinic semialdehyde;
1294
SSAD, SSA dehydrogenase; sucCoA-syn, succinyl CoA synthetase; ThPP, thiamine
1295
pyrophosphate; TIMs, import inner membrane translocases; TOMs, outer membrane
1296
translocases; TPC, thiamine pyrophosphate carrier; tRNAs, tRNA synthetases TXNRD,
1297
thioredoxin reductase; TX-SS, thioredoxin oxidized; TX-SH, thioredoxin reduced;
1298
UCP, uncoupling protein; VDAC, voltage-dependent anion channel
1299
Figure 7: Schematic representation showing the proteins identified by GeLC-MS/MS
1300
related to the functionality of mitochondrial respiratory chain complexes.
1301
Abbreviations: AOX, alternative oxidase; ATPase, ATP synthase; CI, complex I; CII,
1302
complex II; CIII; complex III; CIV, complex IV; CV, complex V; Cyt b, cytochrome b;
1303
Cyt c, cytochrome c; Cyt c1, cytochrome c1; COX, cytochrome oxidase; Ddh,
1304
dihydroorotate dehydrogenase; ETF, electron transfer flavoprotein; exNDH, external
1305
NADH-ubiquinone oxidoreductase; FeS, succinate dehydrogenase iron sulfur subunit ;
1306
GAPdh, glyceraldehydes-3-phosphate dehydrogenase, GLDH, L-galactono-1,4-lactone
1307
dehydrogenase; NAD-DH, NADH dehydrogenase [ubiquinone] subcomplex; Succ DH,
1308
succinate dehydrogenase; UCP, uncoupling protein; UQox, ubiquinone oxidized;
1309
UQred, ubiquinone reduced
1310
Figure 8: Mitochondrial localization prediction (by iPSORT, MitoProt II, Predotar,
1311
TargetP and WoLF PSORT). Predicted to be mitochondrial by one (+), two (++), three
49
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
1312
(+++), four (++++) or all five (+++++) programs. Note that about 35% of the
1313
mitochondrial proteome (both potato and Arabidopsis) are not predicted to be
1314
mitochondrial by any of the five prediction programs. Combination details of the
1315
predictions are given in Supplemental Table S4.
1316
Figure 9: Post-translational modifications (PTMs) in the potato mitochondrial proteome.
1317
The list of PTM analyzed can be found in Table II. Of the 1060 potato mitochondrial
1318
protein groups, 559 (53%) had at least one PTM. Methionine oxidation followed by
1319
proline and lysine oxidation are the most abundant types of PTM. Individual proteins
1320
contained up to nine types of PTMs (Table S6).
1321
50
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
1322
Table I: Functional distribution of the mitochondrial proteins from potato and
1323
Arabidopsis based on the functional classification made by Heazlewood et al. (2004).
1324
The relative frequency (%) of proteins classified in each functional category in the
1325
Arabidopsis and potato proteome are shown. The fold change in protein numbers
1326
(potato/Arabidopsis) are in brackets. The frequency of the relative protein abundance
1327
(% dNSAF) of each functional category in the potato proteome is also shown.
Arabidopsis
Potato
% dNSAF
Function
# proteins
%
# proteins
%
Energy
98
23.6
125
(1.3) 11.8
11.5
Metabolism
81
19.5
216
(2.6) 20.4
21.1
Protein fate
53
12.7
102
(1.9)
9.6
9.3
DNA synthesis and processing
9
2.2
23
(2.6)
2.2
1.8
Transcription
8
1.9
13
(1.6)
1.2
0.9
RNA processing
14
3.4
104
(7.4)
9.8
11.1
Protein synthesis
15
3.6
126
(8.4) 11.9
12.0
Defense, stress, detoxification
16
3.8
30
(1.9)
2.8
2.8
Communication/signaling
19
4.6
39
(2.1)
3.7
3.8
Transport
19
4.6
59
(3.5)
5.6
5.1
Miscellaneous
4
1.0
0
-
0.0
0
Structural organization
9
2.2
5
(0.6)
0.5
0.3
Unknown
71
17.1
218
(3.1) 20.6
20.2
Total
416
1060 (2.5)
1328
1329
1330
51
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Mitochondrial proteome analysis
1. Isolation of mitochondria
2. Protein extraction and SDS-PAGE
fractionation
Percoll gradients
Potato tubers
Potato tuber cells
6. Spectral counting quantification
(dNSAF)k =
Three-step gradient
Continuous gradient
(SAF)k
Ʃ (SAF)n
40 slices are excised from each gel lane
4. LC-MS/MS analysis
5. Database searching
Correlation analysis
PROLUCID
MSGF-DB
SEQUEST
LTQ Orbitrap XL Thermo mass
spectrometer
Experimental MS/MS spectra
MASCOT
Combination of results from
different search engines
Theoretical MS/MS spectra
Mass spectra collected
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
3. In gel digestion
Figure 2: Number of peptides (A) and proteins (B) identified using different database searching
engines <1% protein FDR identification. The total number of non-redundant peptides and proteins
were 7346 and 1060, respectively.
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Figure 3
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Figure 4: Properties of the potato mitochondrial proteome (n = 1060) as compared to
the Arabidopsis mitochondrial proteome (n = 426; [Heazlewood et al. 2004]). (A)
Distribution of molecular mass (kDa), (B) isoelectric point and (C) Grand Average
Hydropathy (GRAVY) score. A disproportionately large number of low-molecularweight proteins with negative Gravy Scores were identified in the potato mitochondrial
proteome).
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Figure 4: Abundance distribution of the potato mitochondrial proteome (Table S2).
Figure 5: Mitochondrial localization prediction results are superimposed on the protein
abundance (spectral counting) [log10(dNSAF)]. Plus signs (+) indicate mitochondrial
localization by as many prediction programs and minus sign (-) indicates localization by
none. Each category on the x-axis corresponds to 0.25 order of magnitude difference in
the abundance of proteins. The difference between the most abundant and the least
abundant proteins was 1778 fold.
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Figure 6: Schematic representation showing proteins that were identified by GeLC-MS-MS in the mitochondrial preparation of potato tubers. Squares
represent protein identification and abundance based on spectral counting. Each color represents a range of Log10 transformed dNSAF (bottom left legend).
Numbers in parentheses represent number of proteins found within the same range of abundance. Respiratory chain complexes (OXPHOS) are showed in
detail in Figure 8. Abbreviations: ABC, ABC transporter; AC, aconitase; ACOT, acyl-coenzyme A thioesterase 9; ACS, Long chain acyl-CoA synthetase
9APX, ascorbate peroxidase; AOX, alternative oxidase; ASC; ascorbate; AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; BCKDH,
branched chain alpha-keto acid dehydrogenase; CAC, carnitine/acylcarnitine carrier protein; CI-CV, respiratory chain complexes (see Figure 7); CoA DHase,
isovaleryl-CoA dehydrogenase; CS, citrate synthase; DiT1, oxoglutarate malate transporter; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase;
DIC, Mitochondrial dicarboxylate carrier protein; D-LDH, D-lactate dehydrogenase; E1α, pyruvate dehydrogenase subunit α; E1β, pyruvate dehydrogenase
subunit β; E2, dihydrolipoamide S-acetyltransferase; E3, Dihydrolipoamide dehydrogenase; ECR, trans-2-enoyl-CoA reductase; EH, Enoyl-CoA hydratase;
ER, enoyl-acyl-carrier-protein reductase; ETF, electron transfer flavoprotein; FCL, 5-formyltetrahydrofolate cycloligase; FDF, formyltetrahydrofolate
deformylase-like; FUM, fumarase; GABA, γ-aminobutyric acid; GABA-T, GABA transaminase; GDC, glycine decarboxylase complex; GDH, glutamate
dehydrogenase; Gly-II, hydroxyacylglutathione hydrolase 3; Gly-I, lactoylglutathione lyase; GPX, glutathione peroxidase; GR, glutathione reductase; GRX,
glutaredoxin; GSH; reduced glutathione; GSSG; oxidized glutathione; GST, glutathione-s-transferase; HAD, hydroxyacyl-thioester dehydratase; HMG lyase,
hydroxymethylglutaryl-CoA lyase; ICDH, isocitrate dehydrogenase; IscA, iron sulfur cluster assembly protein; MCAT, acyl-carrier-protein Smalonyltransferase; MCC, 3-methylcrotonyl-CoA carboxylase; MCD, malonyl CoA decarboxylase; MCP, mitochondrial carrier proteins; MG hydratase,
methylglutaconyl-CoA hydratase; MDH, malate dehydrogenase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase; MFT,
mitochondrial folate transporter/carrier-like; MnSOD, superoxide dismutase; MPP, mitochondrial processing peptidase; NAD-ME, NAD-dependent malic
enzyme; NDT, nicotinamide adenine dinucleotide transporter; 2-OGDH, 2-oxoglutarate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PGP,
phospholipid hydroperoxide glutathione peroxidase; PHB, prohibitin; Pic, phosphate carrier protein; PRDX, peroxiredoxin; PSP, presequence protease; SAM,
S-adenosylmethionine; SAM-T, S-adenosylmethionine-dependent methyltransferase; SAMC, S-adenosylmethionine carrier protein; SDH, succinate
dehydrogenase; SCaMC, calcium-binding mitochondrial carrier protein; SFC, Succinate/fumarate mitochondrial transporter; SHMT, serine
hydroxymethyltransferase; SSA, succinic semialdehyde; SSAD, SSA dehydrogenase; sucCoA-syn, succinyl CoA synthetase; ThPP, thiamine pyrophosphate;
TIMs, import inner membrane translocases; TOMs, outer membrane translocases; TPC, thiamine pyrophosphate carrier; tRNAs, tRNA synthetases TXNRD,
thioredoxin reductase; TX-SS, thioredoxin oxidized; TX-SH, thioredoxin reduced; UCP, uncoupling protein; VDAC, voltage-dependent anion channel.
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Figure 7: Schematic representation showing the proteins identified by GeLC-MS/MS related to the functionality of mitochondrial respiratory chain complex.
Abbreviations: AOX, alternative oxidase; ATPase, ATP synthase; CI, complex I; CII, complex II; CIII; complex III; CIV, complex IV; CV, complex V; Cyt b,
cytochrome b; Cyt c, cytochrome c; Cyt c1, cytochrome c1; COX, cytochrome oxidase; Ddh, dihydroorotate dehydrogenase; ETF, electron transfer flavoprotein;
exNDH, external NADH-ubiquinone oxidoreductase; FeS, succinate dehydrogenase iron sulfur subunit ; GAPdh, Glyceraldehyde 3-phosphate dehydrogenase,
GLDH, L-galactono-1,4-lactone dehydrogenase; NAD-DH, NADH dehydrogenase [ubiquinone] subcomplex; Succ DH, succinate dehydrogenase; UCP,
uncoupling protein; UQox, ubiquinone oxidized; UQred, ubiquinone reduced.
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Figure 8: Mitochondrial localization prediction (by iPSORT, MitoProt II, Predotar,
TargetP and WoLF PSORT). Predicted to be mitochondrial by one (+), two (++), three
(+++), four (++++) or all five (+++++) programs. Note that about 35% of the
mitochondrial proteome (both potato and Arabidopsis) are not predicted to be
mitochondrial by any of the five prediction programs. Combination details of the
predictions are given in the Supplemental TableS3.
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Figure 9: Post-translational modifications (PTMs) in the potato mitochondrial proteome.
The list of PTM analyzed can be found in Table 3. Of the 1060 potato mitochondrial
protein groups, 559 (53%) had at least one PTM. Methionine oxidation followed by
proline and lysineoxidation are the most abundant types of PTM. Individual proteins
contained up to nine types of PTMs (TableS6)
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