multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the...

Multiple Lines of Evidence Localize Signaling,Morphology, and Lipid Biosynthesis Machinery to theMitochondrial Outer Membrane of Arabidopsis[W][OA]

Owen Duncan, Nicolas L. Taylor, Chris Carrie, Holger Eubel, Szymon Kubiszewski-Jakubiak, Botao Zhang,Reena Narsai A. Harvey Millar, and James Whelan*

Australian Research Council Centre of Excellence in Plant Energy Biology (O.D., N.L.T., C.C., H.E., S.K.-J.,B.Z., R.N., A.H.M., J.W.), Centre for Comparative Analysis of Biomolecular Networks (N.L.T., A.H.M.), andCentre for Computational Systems Biology (R.N.), University of Western Australia, Crawley, WesternAustralia 6009, Australia

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, andbiochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane andendoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsisthaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with anumber of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which werenovel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novelfunctions for the plant outer mitochondrial membrane. The novel components identified included proteins that affectedmitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outermembrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknownfunction. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in theouter membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements ofthe mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in thecontext of mitochondrial intracellular interaction.

Mitochondria are double membrane-bound organ-elles. While the inner membrane and its role in oxida-tive phosphorylation have been extensively studied(Eubel et al., 2004), the outer membrane is oftenoverlooked and has been only lightly studied inplants. Much of what is known about the functionsof the outer membrane has been inferred from studiescarried out in yeast (Saccharomyces cerevisiae) and hu-man (Homo sapiens). While this approach has beensuccessful in determining the molecular identities ofseveral key functions of the mitochondrial outer mem-brane, such as the protein import pore Translocase ofthe Outer Membrane 40-kD subunit (TOM40; Janschet al., 1998), and the regulators of mitochondrial dis-tribution and morphology, mitochondrial r-typeGTPases (MIRO; Yamaoka and Leaver, 2008), the lineage-specific evolution and specialization of mitochondrial

function has limited the applicability of much of thedata gathered in other species. For example, only twoof the six components making up the TOM complexin plants, TOM40 and TOM7, have been identified inplant genomes on the basis of sequence identity(Werhahn et al., 2001). Also, outer mitochondrialmembrane proteins, such as OM64, which exhibitssequence similarity to a well-characterized plastid-localized protein, would be missed or incorrectlyannotated in an orthology-based approach comparingmitochondrial proteins from other organisms (Chewet al., 2004).

Using GFP tagging, mitochondria have been visu-alized to undergo fusion, fission, and rapid move-ments, suggesting a dynamic interaction withcomponents of the cytoskeleton (Sheahan et al., 2004,2005; Logan, 2010). However, many of the specificproteins that mediate such processes remain un-known. While mitochondria do play a central role inprogrammed cell death in plants, it is clear that,compared with mammalian systems, different proteincomponents mediate this process. For example, plantgenomes do not appear to have genes encoding theB-cell lymphoma 2 family of proteins, which are knownto mediate mitochondrial membrane permeabilityshifts (Reape and McCabe, 2010). The outer mem-brane not only acts as a barrier for molecules from the

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:James Whelan ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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cytosol to enter mitochondria, it also acts as a barrierfor molecules to leave mitochondria. Thus, the outermitochondrial membrane may also contain proteinsinvolved in signal transduction and mediating retro-grade signals from the mitochondrion to the nucleusor even from the mitochondrion to the plastid. Pro-teins such as Nuclear Control of ATPase (Camougrandet al., 1995) in yeast and the mammalian Mitochon-drial Antiviral Signaling Protein (Koshiba et al.,2011b) are examples of such outer mitochondrialmembrane signaling components identified in otherspecies, none of which have thus far been localizedin plants.

The identification of proteins localized to the mito-chondrial outer membrane is complicated by its rela-tively low protein content and its close associationwith the protein-rich inner membrane and endoplas-mic reticulum. Furthermore, in plants, it has beenshown that the inner and outer mitochondrial mem-branes are physically linked by a C-terminal extensionof the Translocase of the Inner Membrane protein,TIM17-2, which is anchored in both membranes(Murcha et al., 2005). Similarly, in yeast, the Mitochon-drial Distribution and Morphology protein, MDM10(an ortholog of which has not been found in plantgenomes), has been shown to link the mitochondrialand endoplasmic reticulum membranes (Kornmannand Walter, 2010). Attempts to isolate pure outermembrane fractions, therefore, are likely to be com-promised by contamination from either of these twostructures. Previous studies aimed at determining theouter membrane proteome by direct analysis of suchfractions have often suffered from either limited cov-erage (30 proteins found, 67% estimated coverage inNeurospora crassa; Schmitt et al., 2006) or an excess ofcontaminants (112 proteins found, many likely con-taminants, 85% estimated coverage in S. cerevisiae;Zahedi et al., 2006). Coverage estimates are calculatedby summing the total numbers of proteins previouslylocalized to the outer membrane and determiningwhat percentage of these are independently identifiedin these studies. In the case of Arabidopsis (Arabidopsisthaliana) and plants in general, obtaining pure outermitochondrial membrane fractions is further compli-cated by the presence of the chloroplast and a greaterdifficulty in obtaining sufficient quantities of mito-chondria necessary to perform suborganelle proteo-mics on low-abundance fractions.

With these challenges in mind, we developed astatistically rigorous quantitative proteomic workflowto provide a qualitative assessment of suborganelleprotein location in Arabidopsis. We then coupled thiswith independent evaluation by biochemical and cellbiology approaches (Millar et al., 2009) in order toconfidently determine components of the outer mito-chondrial membrane proteome of Arabidopsis. First,we enriched outer membrane vesicles from highlypurified mitochondria and compared the abundanceof its constituents with that in prefractionated “con-taminant” samples. The proteins found to be enriched

in the outer membrane fractions were confirmed ordiscarded as bona fide outer membrane proteins basedon investigation through the literature and furtherexperimental confirmation by GFP tagging and tran-sient expression or by in vitro import of [35S]Met-labeled precursor proteins into purified mitochondria.By hierarchical evaluation of these data, we identified42 mitochondrial outer membrane proteins, 27 ofwhich are novel to this localization, for an estimated88% coverage. The proteins identified range fromplant-specific proteins with unknown functions toproteins that have putative functions in mitochondrialsignaling, morphology, and defense responses.

RESULTS

In order to identify protein components of the mito-chondrial outer membrane, we developed a workflowthat used multiple lines of independent evidence todiscover and confirm the subcellular localizationof proteins on the mitochondrial outer membrane.The first aspect of this workflow was the selection ofmultiple, independent prefractionated samples forcomparisons with enriched mitochondrial outer mem-branes in order to independently analyze the likelycauses of contaminants (Fig. 1A). Furthermore, wesought a control for the protein composition of othermitochondrial membranes (IM), a control for thecoenrichment of contaminating proteineous structuresin the outer membrane isolation process (high-speedpellet-outer membrane [HSP-OM]), and a control fornonmembrane proteins associating with the outermembrane during the cell fraction procedures (mito-plasts). By comparing the abundance of the proteinspresent in each of these fractions (Fig. 1B), we wereable, in effect, to subtract contaminants from themitochondrial outer membrane fraction in a statisti-cally rigorous fashion (Fig. 1C). It should be noted thatthe success or failure of this technique hinges not onthe purity of the fractions analyzed but on the relativeenrichment of sources of contamination in the pre-fractionated samples to levels greater than that ob-served in the mitochondrial outer membrane fraction.The result of these analyses was termed the putativeouter mitochondrial membrane proteome. The puta-tive subcellular/submitochondrial location for each ofthese proteins was then assessed by techniques inde-pendent of the fractionation procedure, including GFPtagging or in vitro mitochondrial protein import ex-periments. To rigorously define the outer mitochon-drial membrane proteome, candidate proteins neededtwo independent pieces of evidence, such as enrich-ment in the outer membrane fraction as determined bymass spectrometry (MS), and confirmation by anotherindependent approach, such as in vitro or in vivotargeting ability to the outer membrane or western-blot analysis (Millar et al., 2009). The application ofthese criteria led to the identification of a stringent setof 42 proteins (Fig. 1C).

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Preparation of Mitochondrial Outer Membrane andPrefractionated Samples

To enrich mitochondria from which outer mem-branes could be isolated, a two-stage separation of theorganelle fraction of Arabidopsis protoplasts wascarried out. This involved first obtaining a crudeorganelle fraction (referred to as the HSP) and thenenriching mitochondria from this fraction, initially bydensity gradient centrifugation and subsequently byfree-flow electrophoresis (FFE). The ability of FFEfractionation to separate mitochondria from othercellular contaminants, even those in close associationwith the mitochondria (such as the endoplasmic retic-ulum) on a basis independent of organelle density (i.e.surface charge) allows mitochondria to be enriched toa greater degree than single physical property isola-tion strategies (Fig. 2). The mitochondrial enrichment

of each of these fractions was assessed by comparisonof Coomassie blue staining profiles (Fig. 2A) andimmunodetection (Fig. 2, B and C) of proteins foundin mitochondria (TOM40), peroxisomes (3-Ketoacyl-Coenzyme A Thiolase [KAT2]), plastids (ribulosebisphosphate carboxylase, large subunit, and light-harvesting complex B [LhcB]), and the endoplasmicreticulum (Calnexin; Fig. 2). Coomassie blue stainingof proteins separated by SDS-PAGE indicated thatboth the gradient-fractionated and FFE-fractionatedsamples were enriched in mitochondria comparedwith the starting material, the HSP (Fig. 2A). Image-based quantitation of band intensity following immu-nodetection confirmed this enrichment, as indicatedby the presence of the outer membrane marker,TOM40-1, which was 2.5-fold higher following gradi-ent fractionation and 3-fold higher after FFE (Fig. 2B).Levels of endoplasmic reticulum were assessed with

Figure 1. Experimental strategy used to determine the outer mitochondrial membrane proteome. Three samples were analyzedto determine a putative outer membrane proteome that was subsequently evaluated by a combination of prior knowledge, GFPlocalization, and in vitro protein uptake experiments. A, Each sample was derived from a crude organelle preparation preparedfrom Arabidopsis cell culture. For the desired fraction (red, mitochondrial outer membrane), isolated mitochondria inhypoosmotic solution were subjected to mechanical disruption. The inner and outer membranes were separated by Suc gradientcentrifugation. Two samples were retained from this procedure: enriched mitochondrial outer membranes and enrichedmitochondrial inner membranes (intramitochondrial contaminants [blue]). Extramitochondrial contaminants (green [HSP-OM])were enriched by subjecting the untreated crude organelle isolation to the same Suc gradient enrichment process used to enrichthe mitochondrial outer membrane. B, Each of these samples was analyzed by MS, and the abundance of constituent proteinswas compared. C, Statistical analysis of the data yielded a putative outer membrane proteome. This putative proteome wasfurther refined by the use of a second quantitative technique, iTRAQ, which included an additional sample designed to eliminatematrix contaminants from the putative outer membrane. Additional evidence from previous literature was used to confirm orreject members of this refined list. Identifications for which no rigorous prior evidence existed were independently confirmed orrejected by the use of GFP localization and/or in vitro protein import experiments.

Arabidopsis Mitochondrial Outer Membrane

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an antibody raised to recombinant human Calreticulin(which primarily detects the membrane-bound formof this protein, Calnexin, in Arabidopsis, as evidencedby the size and solubility of the detected protein).These images indicated that endoplasmic reticulumlevels were reduced 14-fold by gradient fractionationand more than 400-fold by the FFE process. Two majorbands were detected in the HSP fraction (Fig. 2B) cor-responding to the two Calnexin isoforms At5g07340.1(61.4 kD) and At5g61790 (60.4 kD). Peroxisomal con-tamination, as indicated by the presence of KAT2, wasreduced by 1.5-fold after gradient fractionation and28-fold following FFE enrichment. Similarly, anti-bodies raised to the Rubisco large subunit showed a4-fold reduction in chloroplast contamination aftergradient purification and a 60-fold reduction follow-ing FFE (Fig. 2B). An additional membrane-boundplastid marker, LhcB, was readily detectable in en-riched plastid samples and present in the HSP fractionbut was undetectable in either of the mitochondria-enriched fractions (Fig. 2C). This high level of en-richment laid a firm foundation for the subsequentidentification of extramitochondrial contaminantspresent in outer membrane fractions.

Mitochondria were then subfractionated in order toseparate the inner and outer membranes. This processis perhaps the most problematic step in the enrichment

of mitochondrial outer membrane, as the inner andouter membranes are physically linked by proteinssuch as TIM17-2 (Murcha et al., 2005). In order toseparate them, mitochondria were placed in hypoos-motic solution, swelling the highly folded inner mem-brane to beyond the capacity of the unfolded outermembrane. This ruptures the outer membrane, whichis then dissociated from the inner membrane by sev-eral gentle strokes with a Potter-Elvehjem homoge-nizer. The protein-rich, dense inner membranes areseparated from the less dense outer membranes bysedimentation and subsequent flotation through Sucgradients. This membrane fractionation was performedon two samples, the first being FFE-enriched mito-chondria, to yield enriched mitochondrial outer mem-brane (Mt OM), and the second being the crudeorganelle enrichment HSP, to yield a contaminatedouter membrane fraction (HSP-OM). Additional pre-fractionated samples consisting predominantly ofmitochondrial inner membranes (Mt IM) and mito-chondrial inner membranes and matrix (mitoplast)were prepared to allow the identification of con-taminants arising from these compartments. Thecomposition of these samples was examined by im-munodetection of proteins characteristic of the endo-plasmic reticulum, plastids, and the mitochondrialinner membrane (Fig. 3). Coomassie Brilliant Blue

Figure 2. Assessment of the enrichment of mitochondria from Arabidopsis suspension cell cultures. A, One-dimensional SDS-PAGE separation of three fractions isolated at different stages of mitochondrial enrichment. HSP is the result of lysing Arabidopsissuspension cell cultures and pelleting the insoluble fraction. Percoll Grad represents mitochondrial enrichment from the HSP byapplication of the insoluble fraction to two serial Percoll density gradient centrifugations, preserving the fractions most enrichedin mitochondria. In order to obtain our most enriched sample, the gradient-enriched mitochondrial fraction was againfractionated by surface charge utilizing FFE, with the fractions most enriched in mitochondria being retained. B, Assessment ofthe enrichment of mitochondrial fractions by western blot. TOM40-1 (At3g20000.1) is a mitochondrial outer membrane marker,KAT2 (At2g33150.1) is a peroxisomal marker, Calnexin (At5g61790.1 and At5g07340.1) is present in the endoplasmic reticulum,and the large subunit of ribulose bisphosphate carboxylase (RbcL; AtCg00490.1) is a soluble plastid protein. C, Assessment ofplastid contamination of mitochondrial fractions by western blot. At plastids, Fraction enriched in plastids from Arabidopsis; Psplastids, fraction enriched in plastids isolated from pea (Pisum sativum). LhcB was located on the plastid thylakoid membrane.

Duncan et al.





staining of the separated fractions revealed differingprotein profiles for each of the fractionated samples(Fig. 3A). Identification of several high-abundancebands on this gel by electrospray ionization-tandemmass spectrometry (ESI-MS/MS) showed that themost abundant band in the mitochondrial outer mem-brane fraction consists of the previously identifiedmitochondrial outer membrane proteins, Voltage-Dependent Anion Channel1 (VDAC1), -2, -3, and -4.Identification of another high-abundance band pre-sent in the prefractionated samples but absent fromthe outer membrane fraction showed it to predomi-nantly consist of a subunit of the b-subunit of ATPsynthase, which is located on the inner mitochondrialmembrane. Details of these identifications can befound in Supplemental Table S7. Immunodetectionof the mitochondrial outer membrane marker TOM40(At3g20000.1) in these same fractions (Fig. 3B) re-vealed strong enrichment of the outer membrane andcorresponding depletion in the prefractionated sam-ples. Detection of TOM40 in the mitoplast and innermembrane samples is a result of the incompleteseparation of outer membrane from inner membraneduring the homogenization process. Conversely, de-tection of the mitochondrial matrix marker Pyruvate

Dehydrogenase subunit E1 (At1g01090.1), the mito-chondrial inner membrane marker cytochrome coxidase subunit 2 (COXII; AtMg00160.1), the endo-plasmic reticulum membrane marker Calnexin, andthe soluble peroxisome marker KAT2 revealed deple-tion of each of these contaminants in the outer mem-brane fraction and enrichment in at least one of theprefractionated samples. Detection of KAT2 in themitoplast sample is a result of residual peroxisomalcontamination of the mitochondrial enrichment pro-cedure and can also be observed in Figure 2B. Owingto the enriched/unenriched sampling strategy used,this residual contamination is accounted for by theinclusion of HSP-OM, which is more enriched inperoxisomes and plastids than Mt OM (Fig. 2B); how-ever, KAT2, being a soluble protein, is lost in thepreparation of this membrane sample. Immunodetec-tion of additional membrane-anchored proteins char-acteristic of the plastid outer envelope (TOC159;At4g02510.1) and peroxisomal membrane (PMP22;At4g04470.1) did not detect these proteins in any ofthe subfractionated samples. However the chloro-plast membrane proteins TOC159 (data not shown)and LhcB (Fig. 2C) were detected in purified chloro-plasts.

Figure 3. Assessment of the enrichment of mitochondrial outer membrane and contaminants. A, One-dimensional SDS-PAGEseparation of contaminant and purified outer membrane fractions. The indicated band identifications were conducted by ESI-MS/MS, and details can be found in Supplemental Table S7. B, Western-blot assessment of the experimental fractions used forsemiquantitative analysis by MS. TOM40-1 (At3g20000.1) is a mitochondrial outer membrane marker, pyruvate dehydrogenasesubunit E1a (PDH E1a) is present in the mitochondrial matrix, COXII (AtMg00160.1) is on the matrix side of the innermitochondrial membrane, Calnexin (At1g56340) is present on the endoplasmic reticulum membrane, and KAT2 (At2g33150) isa soluble peroxisomal marker. These membranes were also probed with the additional antibodies TOC159, TOC75, translocaseof inner chloroplast membrane 40-kD subunit (TIC40), and peroxisomal membrane protein 22, but each of these antibodiesfailed to detect appropriately sized products.






MS Analysis of Outer Membrane Composition

The first quantitative approach undertaken in-volved the counting of spectra that matched individ-ual proteins in the purified outer membrane fraction.These counts were then comparedwith those observedin the prefractionated samples. The first of thesecomparisons was designed to eliminate membrane-associated contaminants arising from other organelles/membrane systems (Mt OM:HSP-OM). The second ofthese comparisons was designed to identify contam-inants in the Mt OM fraction that arose from othermitochondrial compartments (Mt OM:Mt IM). Thesecomparisons enabled a quantitative assessment ofwhether their constituent proteins were located on theouter mitochondrial membrane or elsewhere in thecell, allowing the assembly of a short list of likely outermembrane proteins.

Each of the protein samples (Mt OM, Mt IM, andHSP-OM) was acetone precipitated, digested, andanalyzed by ESI-MS/MS. A total of 751 unique Arabi-dopsis proteins were identified across three biologicalreplicates of this set of samples. A false discovery ratefor this data set was empirically determined by com-piling the mass spectra from each of the ESI-MS/MSanalyses and searching this file against a decoy (shuf-fled The Arabidopsis Information Resource [TAIR] 9data set) database. These searches indicated a falsediscovery rate of 1.5% at the peptide level. Of the 751proteins, 185 were identified in the outer membranefraction (Mt OM) in more than one biological replicate(Supplemental Table S1).

By modeling the numbers of spectra observed foreach protein using Poisson regression, we found atotal of 64 proteins that were enriched in the outermembrane fractions using the following criteria: mustbe detected in two or more biological replicates andhave a P value of 0.13 or less for enrichment over boththe HSP-OM and Mt IM samples when the spectrafrom all biological replicates are pooled (SupplementalTable S2). This P value was intentionally less stringentat this preliminary stage in order to maximize theability of these analyses to discover putative mito-chondrial outer membrane proteins, given that anyunintentionally included contaminants would be re-moved in the subsequent experimental confirmationsteps. The validity of this analysis was also investi-gated by GFP-targeting studies of several proteinsfalling on either side of this cutoff, which indicatedthat loosening the P value further would increase thenumber of contaminants without includingmore outermembrane proteins in the preliminary list (data notshown). Proteins that were only detected in theMt OMsamples, provided that they were present in more thanone biological replicate, were also included on thebasis that these were likely to be lower abundance mi-tochondrial outer membrane proteins, only detectedfollowing enrichment.

This list of 64 proteins did not contain abundantmitochondrial inner membrane proteins such as the

ADP/ATP carrier and was generally devoid of com-ponents of the mitochondrial respiratory chain. The 64proteins encompassed 15 of the 17 proteins that havepreviously been shown to be located in the outermitochondrial membrane in various studies in plants(Werhahn et al., 2001; Chew et al., 2004; Scott et al.,2006; Lister et al., 2007; Xu et al., 2008; Yamaoka andLeaver, 2008; Lee et al., 2009). Despite the difficultiesinherent in working with plant cells, such as limitedstarting material, lysis complications due to the cellwall, and extra contamination arising from an addi-tional organelle, the percentage of proteins in thispreliminary list compares favorably with previouslypublished outer mitochondrial membrane proteomesfrom nonplant species. We calculated 88% coverage ofknown outer membrane mitochondrial proteins in ourpreliminary data set versus 85% in S. cerevisiae (Zahediet al., 2006) and 67% in N. crassa (Schmitt et al., 2006).This coverage was achieved with a pool of just 64proteins versus 118 in S. cerevisiae. The two proteinsthat were not included in this preliminary set wereTOM7 (At5g41685.1) and BIGYIN (At3g57090.1; anArabidopsis ortholog of Fis1 in S. cerevisiae and mam-mals; Scott et al., 2006).

Literature, MS, GFP Fluorescence, and in Vitro Import toConfirm Outer Membrane Composition

Additional lines of evidence were sought until eachprotein present in the 64-member putative outer mem-brane proteome had two independent lines of evi-dence to confirm this location or a single high-qualityline of evidence to dispute its inclusion in this list. Asummary of this information can be found in Tables Iand II and Supplemental Table S3.

Literature

Components of the TOM complex have been iso-lated previously from mitochondrial outer membranepreparations and identified by a variety of methods(Werhahn et al., 2001; Braun et al., 2003), contributingliterature evidence for the TOM components TOM 40-1,20-2, 20-3, 20-4, 9-1, 9-2, 5, and 6. Other members ofthe mitochondrial protein import apparatus, Metaxin(At2g19080.1) and OM64 (At5g09420.1), have previ-ously been shown to be located in the outer mitochon-drial membrane by GFP studies (Chew et al., 2004;Lister et al., 2007). Several members of the VDACfamily, DGS1 (At5g12290.1) and ELM1 (At5g22350.1),have also been shown to be located in the mitochon-drial outer membrane by GFP studies (Arimura et al.,2008; Xu et al., 2008; Lee et al., 2009; Table I). A numberof proteins present in the putative outer mitochondrialmembrane proteome in this study have previouslybeen reported to be in other subcellular locations.These proteins included TIM9 (At3g46560.1) andTIM13 (At1g61570.1), which have been previouslyreported to be soluble proteins of the mitochondrialintermembrane space (Koehler, 2004; Lister et al.,

Duncan et al.





Table I. Proteins confirmed to be in the mitochondrial outer membrane

Columns from left to right are as follows: AGI No., Arabidopsis Genome Initiative identifier (www.Arabidopsis.org); Description, commonidentifier; Raw Counts, tally of the total number of identifying ions summed from three biological replicates from the outer membrane samplefollowed by the HSP-OM and then the inner membrane sample; Ratio OM:HSP-OM, number of identifying ions identified in the outer membranesample divided by the number found in the HSP-OM sample; P, P value applying to the magnitude of change (Supplemental Tables S3 and S4) ascalculated using Poisson regression analysis; Ratio OM:IM, number of identifying ions identified in the outer membrane sample divided by thenumber found in the inner membrane sample; P, P value applying to the magnitude of change (Supplemental Tables S3 and S4) as calculated usingPoisson regression analysis; iTRAQ, these three columns contain the ratios of reporter ions in IM/OM (inner membrane divided by outer membrane),Whole/OM (whole mitoplasts [mitochondria with outer membrane ruptured but inner membrane intact] divided by outer membrane HSP-OM/OM[HSP-OM divided by outer membrane]; values reported here are significant according to the criteria detailed in “Results”); GFP Location,interpretation of subcellular localization resulting from fluorescence microscopy (ÖMtOM refers to a sole mitochondrial outer membranelocalization, while ÖMtOM++ refers to a localization on the mitochondrial outer membrane and other locations); Reference, references topreviously published location information. A single protein, At5g05520.1 (SAM50-2), demonstrated enrichment in the mitochondrial outermembrane fractions but did not display specific targeting when tagged with GFP; it is included here due to evidence gathered from protease-treatedmitochondria (Fig. 5). ‘ is used to indicate that the ratio cannot be calculated because a null value (0) was obtained and thus the P value cannot becalculated, as shown by ^.

AGI No. Description

Raw Counts Spectral Counting iTRAQGFP

LocationReferenceOM:HSP-

OM:IM

Ratio

OM:HSP-OMP

Ratio

OM:IMP IM/OM Whole/OM HSP-OM/OM

At1g04070.1 TOM9-2 3:0:0 ‘ ^ ‘ ^ Werhahn

et al. (2001)

At1g05270.1 TraB family

protein

41:7:0 5.9 1.50E-05 ‘ ^ ÖMtOM

At1g06530.1 Myosin related 53:13:0 4 5.60E-06 ‘ ^ 0.285 0.462 ÖMtOM

At1g24267.1 Unknown protein 13:3:0 4.3 0.022 ‘ ^ ÖMtOM

At1g27390.1 TOM20-2 6:0:0 ‘ ^ ‘ ^ Werhahn

et al. (2001)

At1g49410.1 TOM6 11:2:0 5.5 0.027 ‘ ^ Werhahn

et al. (2001)

At1g50460.1 HXKL-1 41:22:0 1.9 0.018 ‘ ^ 0.165 ÖMtOM

At1g53000.1 KDSB 53:24:16 2.2 0.001 3.3 0.00003 0.305 0.229 ÖMtOM

At2g01460.1 ATP

binding/kinase

8:0:0 ‘ ^ ‘ ^ ÖMtOM

At2g19080.1 Metaxin 14:6:1 2.3 0.082 14 0.011 Lister

et al. (2007)

At2g19860.1 HXK2 92:71:25 1.3 0.101 3.7 8.00E-09 0.276 0.255 0.264 ÖMtOM

At2g38280.1 FAC1 7:2:0 3.5 0.118 ‘ ^ ÖMtOM

At2g38670.1 PECT1 55:28:0 2 0.004 ‘ ^ 0.233 0.417 ÖMtOM

At3g01280.1 VDAC1 90:50:53 1.8 0.001 1.7 0.002 0.27 0.254 0.31 Lee

et al. (2009)

At3g11070.1 SAM50-1 53:24:3 2.2 0.001 17.7 1.30E-06 0.564 0.511 0.429 ÖMtOM

At3g20000.1 TOM40-1 93:42:12 2.2 1.90E-05 7.7 2.4E-11 0.217 0.274 0.395 Werhahn

et al. (2001)

At3g20040.1 HXK4 13:4:0 3.2 0.039 ‘ ^ ÖMtOM

At3g27080.1 TOM20-3 23:0:0 ‘ ^ ‘ ^ Werhahn

et al. (2001)

At3g27930.1 Unknown

protein

44:13:0 3.4 0.0001 ‘ ^ 0.162 0.231 ÖMtOM

At3g50930.1 BCS-1 29:13:0 2.2 0.016 ‘ ^ ÖMtOM

At3g50940.1 BCS-1-like 10:4:0 2.5 0.121 ‘ ^ ÖMtOM

At3g58840.1 Unknown

protein

30:5:0 6 0.0002 ‘ ^ ÖMtOM

At3g63150.1 MIRO2 7:0:0 ‘ ^ ‘ ^ ÖMtOM

At4g29130.1 HXK1 124:97:31 1.3 0.07 4 1.2E-12 0.213 0.23 ÖMtOM

At5g05520.1 SAM50-2 32:8:13 4 0.0004 2.5 0.006 See legend

At5g08040.1 TOM5 21:12:8 1.75 0.122 2.6 0.02 Werhahn

et al. (2001)

At5g09420.1 OM64 34:13:0 2.6 0.003 ‘ ^ 0.203 0.211 0.195 Chew

et al. (2004)

At5g12290.1 DGS1 72:23:3 3.2 1.9E-06 24 7E-08 0.446 Xu et al. (2008)

At5g15090.1 VDAC3 97:63:63 1.5 0.008 1.5 0.008 0.201 0.185 0.234 Lee et al. (2009)

At5g17770.1 NADH:cytochrome

B5 reductase

34:7:0 4.9 0.0001 ‘ ^ 0.422 0.377 0.372 ÖMtOM

At5g22350.1 ELM1 17:6:0 2.8 0.028 ‘ ^ ÖMtOM Arimura

et al. (2008)

At5g27540.1 MIRO1 89:55:8 1.6 0.005 11.1 7E-11 0.2 0.214

At5g40930.1 TOM20-4 8:0:0 ‘ ^ ‘ ^ Werhahn

et al. (2001)

At5g43970.1 TOM9-1 15:5:4 3 0.033 3.75 0.019 Werhahn

et al. (2001)

(Table continues on following page.)






2005). A variety of other proteins in the set of 64 areknown to be proteins in the inner mitochondrialmembrane: Ubiquinone cytochrome c oxidoreduc-tase-like family protein (At3g52730.1) is a subunit ofcomplex III (Meyer et al., 2008), F0-ATPase subunit 9(AtMg01080.1) has been shown to be a member ofcomplex V, and Frostbite1 (FRO1; At5g67590.1), un-known protein (At1g68680.1), and unknown protein(At4g16450.1) are subunits of complex I (Meyer et al.,2008). Isocitrate Dehydrogenase1 (IDH1; At4g35260.1)has also been shown to be present in the inner mem-brane/matrix fraction of mitochondria (Zhao andMcAlister-Henn, 1996; Table II).

MS-iTRAQ

Initial inspection of the putative proteome indicatedthe presence of soluble contaminants arising from themitochondrial matrix. IDH6 (At3g09810.1) and Aspar-tate Aminotransferase (ASP1; At2g30970.1; Table II)are two high-abundance matrix enzymes. We chose toaddress this source of contamination by conducting anadditional quantitative proteomic study using isobarictags. This study evaluated the abundance of proteinsin four fractions: the same three as used in the spectralcounting analysis with the addition of a sample com-posed chiefly of mitoplasts (i.e. mitochondrial matrixand inner membrane structures depleted in outermembrane). A ratio of 0.7 at P , 0.05 was selected asthe cutoff on the basis that known outer membraneproteins were included, while known contaminantswere excluded. The validity of this cutoff was con-firmed by GFP localization of IDH6 (iTRAQ [forisobaric tags for relative and absolute quantitation]mitoplast:outer membrane ratio of 0.8, geometric SD of1.176) and g-aminobutyrate (GABA) aminotransferase(At3g22200.1; iTRAQ mitoplast:outer membrane ratioof 0.776, geometric SD of 1.185), two proteins that fellclose to but outside the cutoff and were shown tolocalize to the mitochondria but not to the outermembrane (Supplemental Fig. S3, I and II). Thus, theiTRAQ analysis using these criteria contributed evi-

dence for the removal of four proteins from the listof 64, namely ASP1, IDH6, GABA aminotransferase,and a putative NADH:cytochrome B5 reductase(At5g20080.1).

GFP Tagging

These exclusions left a group of 38 proteins forwhich additional evidence was sought in the form ofGFP localization. To accurately use this method, it wasfirst established that GFP and fluorescence microscopywere capable of distinguishing mitochondrial innermembrane or matrix proteins from those located onthe outer mitochondrial membrane (Fig. 4). Forthese purposes, an inner membrane protein, FRO1(At5g67590.1; Heazlewood et al., 2003), a matrix pro-tein, IDH6 (At3g09810.1; Heazlewood et al., 2004),and a previously characterized outer membrane pro-tein, Elongated Mitochondria1 (ELM1; At5g22350.1;Arimura et al., 2008), fused to GFP, were transientlytransformed into Arabidopsis cell culture along with amitochondrial matrix-targeted red fluorescent proteincontrol and observed using fluorescence microscopy(Fig. 4). A clear difference was observed between thefluorescence patterns of the control proteins located inthe inner membrane-FRO1 (Fig. 4A) and matrix-IDH6(Fig. 4B) and the outer membrane-ELM1 (Fig. 4C). Theinner membrane- and matrix-located controls showedfilled, green, circular fluorescence patterns, whereasthe outer membrane control (ELM1) displayed greenring patterns. This ring pattern can also be observedfor the unknown function At5g55610.1 (Fig. 4D),which was in the set of 38 and deemed to be a positivelocalization of the GFP fusion protein to the mitochon-drial outer membrane. This is also consistent withprevious reports using GFP tagging of mitochondrialouter membrane proteins (Setoguchi et al., 2006; Listeret al., 2007). Similar ring structures corresponding toa mitochondrial outer membrane location were ob-served for 25 of the 38 proteins tested. Of these 25proteins, 21 only displayed this pattern surroundingthe red fluorescent protein control (Supplemental Fig.

Table I. (Continued from previous page.)

AGI No. Description

Raw Counts Spectral Counting iTRAQGFP

LocationReferenceOM:HSP-

OM:IM

Ratio

OM:HSP-OMP

Ratio

OM:IMP IM/OM Whole/OM HSP-OM/OM

At5g55610.1 Unknown

protein

50:19:0 2.6 0.0003 ‘ ^ ÖMtOM

At5g57490.1 VDAC4 80:36:37 2.2 0.00007 2.2 0.0001 0.187 0.249 0.199 Lee et al. (2009)

At5g60730.1 Anion transport 13:5:0 2.6 0.07 ‘ ^ ÖMtOM

At5g67500.1 VDAC2 122:77:64 1.6 0.002 1.9 0.00003 Lee et al. (2009)

Outer membrane

and other

At5g16870.1 Aminoacyl-tRNA

hydrolase

16:6:0 2.7 0.04 ‘ ^ ÖMtOM++

At5g20520.1 WAV2 16:3:0 5.3 0.008 ‘ ^ ÖMtOM++

At4g35970.1 APX5 10:4:0 2.5 0.121 ‘ ^ ÖMtOM++

At5g39410.1 Unknown

protein

52:34:3 1.5 0.054 17.3 1.5E-06 0.464 ÖMtOM++

Duncan et al.





Tab

leII.Proteinsfoundnotto

bein

themitoch

ondrial

outermem

brane

From

theinitiallistof64proteinsfoundto

been

rich

edin

theoutermem

branesample,22wereex

cluded

from

theoutermem

braneproteomebyoneormore

lines

ofad

ditional

eviden

ce.

Columnsfrom

leftto

righ

tareas

follows:Lo

cation,thesubce

llularco

mpartm

entalizationinterpretation;AGINo.,ArabidopsisGen

omeInitiative

iden

tifier

(www.Arabidopsis.org);Description,

commoniden

tifier;Raw

Counts,tallyofthetotalnumber

ofiden

tifyingionssummed

from

threebiologica

lreplicatesfrom

theoutermem

branesample

followed

bytheHSP

-OM

andthen

the

inner

mem

branesample;Ratio

OM:H

SP-O

M,number

ofiden

tifyingionsiden

tified

intheoutermem

branesample

divided

bythenumber

foundin

theHSP

-OM

sample;P,Pvalueap

plyingto

themag

nitudeofch

ange

(Supplemen

talTablesS1

andS2

)as

calculatedusingPo

issonregressionan

alysis;Ratio

OM:IM,number

ofiden

tifyingionsiden

tified

intheoutermem

branesample

divided

bythenumber

foundin

theinner

mem

branesample;P,Pvalueap

plyingto

themag

nitudeofch

ange

(Supplemen

talTablesS1

andS2

)as

calculatedusingPoissonregressionan

alysis;

iTRAQ,thesethreeco

lumnsco

ntain

theratiosofreporter

ionsin

IM/O

M(inner

mem

branedivided

byoutermem

brane),W

hole/O

M(w

hole

mitoplasts[m

itoch

ondriawithoutermem

brane

rupturedbutinner

mem

braneintact]divided

byoutermem

braneHSP

-OM/O

M[H

SP-O

Mdivided

byoutermem

brane];values

reported

herearesign

ifica

ntac

cordingto

thecriteria

detailedin

“Results”);GFP

Location,interpretationofsubce

llularloca

liza

tionresultingfrom

fluorescen

cemicroscopy;

InVitro

ImportResult,mitoch

ondrial

loca

lizationoftested

proteinsas

determined

by

relian

ceonthepresence

ofan

inner

mem

braneelec

troch

emical

grad

ient;PreviousPublished

Location,loca

tionofpreviouslystudiedproteins;Referen

ce,referencesto

previouslypublished

loca

tioninform

ation.Th

efinal

protein

inthis

table,At4g1

7140.2,was

foundto

beab

undan

tan

dstronglyen

rich

edin

themitoch

ondrial

outermem

branesamples;

however,co

nfirm

ationof

loca

liza

tionindep

enden

toftheorgan

elle

frac

tionationwas

attemptedbutnotobtained

.‘isusedto

indicatethat

theratioca

nnotbeca

lculatedbecau

seanullvalue(0)was

obtained

andthusthe

Pvalueca

nnotbeca

lculated,as

shownby^.

Loca

tion

AGINo.

Description

Raw

Counts

Spectral

Counting

iTRAQ

GFP

Location

InVitro

ImportResult

Previous

Published

Loca

tion

Referen

ceOM:H

SP-O

M:

IMRatio

OM:H

SP-O

MP

Ratio

OM:IM

PIM

/

OM

Whole/

OM

CON/

OM

Mitoch

ondrial

At2g3

0970.1

ASP

123:0:9

‘^

2.6

0.017

1.229

1.284

Mitoch

ondrial

At5g2

0080.1

NADH-cytoch

rome

B5reductase,

putative

30:9:4

3.3

0.002

7.5

0.00015

0.713

0.611

Inner

mem

braneormatrix

Notmitoch

ondrial

At1g7

5200.1

Flavodoxin

family

protein

22:7:0

3.1

0.008

‘^

Not mitoch

ondrial

Notmitoch

ondrial

At2g2

6240.1

Unkn

own

protein

9:0:0

‘^

‘^

Not mitoch

ondrial

Notmitoch

ondrial

At2g4

5870.1

Bestrophin-

like

protein

4:0:0

‘^

‘^

Not mitoch

ondrial

Notmitoch

ondrial

At3g6

3520.1

CCD1

40:20:2

20.011

20

3.5E-05

0.341

0.51

Not mitoch

ondrial

Notmitoch

ondrial

At4g0

0355.1

Unkn

own

protein

8:0:0

‘^

‘^

Not mitoch

ondrial

Notmitoch

ondrial

At4g2

7760.1

FEY

4:0:0

‘^

‘^

Not mitoch

ondrial

Notmitoch

ondrial

At4g3

3360.1

Terpen

e

cyclase/mutase-

related

10:0:0

‘^

‘^

Not mitoch

ondrial

Mitoch

ondrial

At5g6

7590.1

FRO1

5:0:0

‘^

‘^

Mitoch

ondrial

Inner mem

brane,

complexI

Meyer

etal.

(2008)

Mitoch

ondrial

At3g0

9810.1

IDH6

17:0:4

‘^

4.2

0.009

0.557

0.802

Mitoch

ondrial

Mitoch

ondrial

At3g2

2200.1

GABA

aminotran

sferase

47:13:29

3.6

0.00004

1.6

0.041

0.776

0.403

Mitoch

ondrial

Inner mem

brane

ormatrix

Mitoch

ondrial

At5g1

5640.1

Mitoch

ondrial

substrate

carrierfamily

protein

7:0:0

‘^

‘^

Mitoch

ondrial

Inner mem

brane

ormatrix

Mitoch

ondrial

At4g0

4180.1

Unkn

own

protein

10:0:0

‘^

‘^

Mitoch

ondrial

Inner mem

brane

ormatrix

Mitoch

ondrial

At1g6

1570.1

TIM

13

14:3:0

4.7

0.015

‘^

Interm

embrane

space

Lister etal.

(2004)

Mitoch

ondrial

At1g6

8680.1

Unkn

own

protein

8:0:0

‘^

‘^

Inner mem

brane,

complexI

Meyer

etal.

(2008)

(Tab

leco

ntinues

onfollowingpage.)






S1), indicating an exclusive mitochondrial outer mem-brane localization, while four of the tested proteinsdisplayed fluorescence patterns consistent with lo-calization to the mitochondrial outer membrane butalso localized to other intracellular structures, whichwere apparently not mitochondrial in nature (Sup-plemental Fig. S2). GFP analysis of the remaining 12proteins showed that seven of these did not local-ize to the mitochondria (Supplemental Fig. S4), twowere seen to be mitochondrial but did not displaythe ring structure characteristic of outer membraneproteins (At5g15640.1 and At4g04180.1; Supplemen-tal Fig. S3, III and IV), and three proteins failed todisplay any GFP-related fluorescence or were incon-sistent from cell to cell. Images of all of the abovefluorescence patterns can be viewed in SupplementalFigures S1 to S4.

In Vitro Uptake Assays

The two mitochondria-localized proteins that didnot display the ring structure (At5g15640.1 andAt4g04180.1) were selected for in vitro protein uptakeexperiments. Both were seen to be protease protectedwhen incubated with mitochondria under conditionsthat support protein uptake (Supplemental Fig. S3, IIIand IV). When valinomycin was added to the in vitrouptake assay, no protease protection was evident,indicating the requirement of membrane potentialfor import that is a feature of inner membrane ormatrix localization (Supplemental Fig. S3, III and IV).

Of the three proteins for which a consistent GFPlocalization could not be confirmed, two were deemedto be outer membrane proteins on the basis that otherisoforms of the protein family had been investigated aspart of this study and were found to be localized to theouter mitochondrial membrane. These proteins wereSAM50-1 (At3g11070.1) and MIRO1 (At5g27540.1).The final protein, the unknown protein (At4g17140.2),was excluded, as no additional evidence was foundfor its localization beyond the quantitative MS data.The N- and C-terminal 200 amino acids of this large(471.2-kD) protein were fused to GFP but failed toshow a consistent subcellular localization. Together,the MS, literature searches, and GFP analysis pro-duced a final list of 42 plant outer membrane proteins,27 of these being novel, doubling the number ofconfirmed outer membrane proteins in plant mito-chondria and identifying all but two proteins that werepreviously known.

Additional Confirmation of Novel Mitochondrial OuterMembrane Localizations

In order to gain further localization confirmation forthe three novel mitochondrial outer membrane pro-teins, 3-deoxy-manno-octulosonate cytidylyltransfer-ase (KDSB; At1g53000.1), phosphorylethanolamineT

able

II.(Continued

from

previouspag

e.)

Location

AGINo.

Description

Raw

Counts

Spec

tral

Counting

iTRAQ

GFP

Location

InVitro

ImportResult

Previous

Published

Location

Referen

ceOM:H

SP-O

M:

IMRatio

OM:H

SP-O

MP

Ratio

OM:IM

PIM

/

OM

Whole/

OM

CON/

OM

Mitoch

ondrial

At3g4

6560.1

TIM

94:0:0

‘^

‘^

Interm

embrane

space

Lister etal.

(2004)

Mitoch

ondrial

At3g5

2730.1

UQCRX-like

familyprotein

8:0:0

‘^

‘^

Inner mem

brane,

complexIII

Meyer

etal.

(2008)

Mitoch

ondrial

At4g1

6450.1

Unkn

own

protein

5:0:0

‘^

‘^

Inner mem

brane,

complexI

Klodman

n

etal.

(2010)

Mitoch

ondrial

AtM

g01080.1

Subunit9of

mitoch

ondrial

F 0-ATPase

4:0:0

‘^

‘^

Inner mem

brane,

complexV

Meyer

etal.

(2008)

Mitoch

ondrial

At4g3

5260.1

IDH1

24:0:6

‘^

40.002

0.486

Mitoch

ondrial

matrix

Zhao

and

McA

lister-

Hen

n

(1996)

Nofurther

eviden

ceAt4g1

7140.2

Unkn

own

protein

220:104:21

2.1

1.5E-07

10.5

6.22E-

27

0.128

0.203

0.32

See legend

Duncan et al.





cytidylyltransferase (PECT1; At2g38670.1), and thesorting and assembly machinery protein SAM50(At3g11070.1), polyclonal antibodies were raised fol-lowing bacterial expression and purification of por-tions of these proteins. Isolated mitochondria weretreated with increasing amounts of proteinase K. Thistreatment degrades portions of mitochondrial proteinsthat were accessible to the protease and preservedproteins that were protected by the lipid bilayer of themitochondrial outer membrane. Treated mitochondriawere separated by SDS-PAGE, and a number of pro-teins were immunodetected. TOM20-3 is a mitochon-drial outer membrane protein that is exposed to thecytosol (Heins and Schmitz, 1996; Fig. 5A). This pro-tein is rapidly degraded at low concentrations ofproteinase K. Examination of KDSB (Fig. 5B) revealeda similar breakdown profile, indicating that part of this

protein is exposed to the cytosol in vivo and is likely tobe located on the outer mitochondrial membrane.PECT1 (Fig. 5C) was also seen to be accessible to theproteinase. Orthologs of Arabidopsis SAM50 havebeen observed to be localized to the outer mitochon-drial membrane in S. cerevisiae (Kozjak et al., 2003).SAM50, like TOM40, is a transmembrane b-barrelprotein descended from the bacterial ancestor proteinOMP85 (Kozjak et al., 2003), and it appears that thesetwo proteins are not readily accessible to the externallyapplied proteinase (Fig. 5, D and E). At the two highestconcentrations of proteinase, both of these proteinswere seen to be partially broken down, whereas theRieske Iron Sulfur Protein (RISP; Fig. 5F), which islocated on the inner mitochondrial membrane, wasresistant to digestion. This indicates that SAM50, inagreement with the other data presented here, is likely

Figure 4. GFP tagging of mitochon-drial outer membrane proteins toconfirm intraorganelle localization.Arabidopsis suspension cell culturewas transiently transformed with mito-chondrial red fluorescent protein (RFP)control and GFP-tagged experimentalproteins. A, FRO1:GFP, inner mito-chondrial membrane protein. B, IDH6:GFP, mitochondrial matrix-located pro-tein. C, ELM1:GFP, outer mitochondrialmembrane protein. D, Example of novelmitochondrial outer membrane pro-tein displaying a similar fluorescencepattern to ELM1, identified by MS andlocation confirmed by fluorescencemicroscopy. E, GFP only.






to be located on the outer mitochondrial membrane inArabidopsis and that mitochondrial integrity was notcompromised by the proteinase treatment.

Transient Overexpression of Outer Membrane ProteinAffects Mitochondrial Morphology and Segregation

An unexpected outcome of using full-length codingsequences fused to GFP was the observation thatseveral of the GFP fusion proteins apparently alteredthe mitochondrial morphology in transformed cells. Inthe case of KDSB (At1g53000.1), highly abnormal giantmitochondria occupied much of the available space in

observed cells (Fig. 6A). This protein appears to be ofbacterial origin (Misaki et al., 2009), is thought to beinvolved in the synthesis of the outer envelope lipo-polysaccharide KDO2-Lipid A, and has been locatedin mitochondria, but not to the outer membrane, inprevious studies in Arabidopsis (Heazlewood et al.,2004). PECT1 is the penultimate enzyme involved inthe biosynthesis of phosphatidylethanolamine, an im-portant membrane phospholipid. The overexpressionof the outer membrane-targeted fusion protein (Fig.6B) apparently causes mitochondria to bunch togetherat one pole of the cell. Overexpression of a mito-chondrial outer membrane NADH:cytochrome B5 re-ductase (Fig. 6C) also appears to alter mitochondrialmorphology when compared with the more typicalmitochondrial outer membrane fluorescence patternseen with the unknown protein At5g55610.1 (Fig. 6D).

Transcriptomic Analysis of Genes Encoding OuterMembrane Proteins Reveals Patterns of Coexpression

In order to determine whether there was a relation-ship between colocalization and coexpression, therelative expression levels for the 38 genes that werepresent on the Affymetrix ATH1 microarray wereanalyzed during germination and across the Arabi-dopsis developmental microarray series (Schmid et al.,2005). Additionally, the patterns of transcript abun-dance for 247 genes that encode inner membraneproteins were analyzed to determine if the transcriptabundance of genes that encode outer membraneproteins, or subsets of outer membrane proteins, dif-fered from the patterns of transcript abundance forgenes that encode inner membrane proteins. Datawere normalized to make them comparable (see“Materials and Methods”), and expression levels aredisplayed relative to maximum expression (Fig. 7;Supplemental Fig. S5). Overall, it was observed thatthe transcript abundance of genes encoding outer andinner membrane mitochondrial proteins displayedmaximum levels during seed germination, with asubset showing relatively high levels in root tissues(Supplemental Fig. S5). When all genes encoding innermembrane- and outer membrane-localized proteinswere clustered together over a germination timecourse, it was seen that genes encoding outer mem-brane proteins were significantly underrepresented incluster 2 (P , 0.05) compared with genes encodinginner membrane proteins (13% outer membrane ver-sus 27% inner membrane; Fig. 7). Gene in cluster 2displayed high transcript abundance in fresh harves-ted seeds that decreased during stratification. In con-trast, 45% of genes encoding outer membrane proteins(versus 21% of inner membrane protein-encodinggenes) were seen in the group of genes showingtransient expression during germination in cluster 4(Fig. 7, boxed in yellow), which represents a significantoverrepresentation (P = 0.0007) of genes encodingouter membrane proteins in this cluster. Genes incluster 4 peak in transcript abundance during the first

Figure 5. Immunodetection of isolated mitochondria treated withproteinase K. A, TOM20-3 is a mitochondrial outer membrane-anchored protein with a large cytosolic domain and as such isvulnerable to digestion by external application of proteinase K. B andC, KDSB (B) and PECT1 (C) are novel outer membrane proteinsidentified in the course of this study by MS and confirmed by GFPlocalization. The breakdown of KDSB is similar to that of TOM20-3,indicating that it is also exposed to the cytoplasm. PECT1 is brokendown by external application of proteinase K but appears to be moreresistant than TOM20 and KDSB. D and E, SAM50 (D) and TOM40 (E)are b-barrel outer membrane proteins and as such are embedded in themembrane, rendering them resistant to digestion. F, RISP is found in theubiquinol-cytochrome c reductase complex in the mitochondrial innermembrane. Its preservation indicates that the integrity of the innermembrane was maintained throughout the proteinase dilution series.

Duncan et al.





24 h of germination and decrease in abundance inyoung seedlings, as under these conditions germina-tion is completed at 24 h in Arabidopsis, as the radiclehas emerged (Weitbrecht et al., 2011). Thus, althoughgenes encoding mitochondrial outer membrane pro-teins follow the general pattern observed for genesencoding inner membrane proteins, in that they dis-play maximum levels of transcript abundance duringseed germination, detailed time-course analysis re-veals that for a large subset (i.e. cluster 4) this differsfrom the majority of genes encoding mitochondrialproteins. Previously, it has been shown for maize (Zeamays) and rice (Oryza sativa) that active mitochondrialbiogenesis precedes the expression of respiratorychain components of the inner membrane (Loganet al., 2001; Howell et al., 2006); thus, the patternobserved here suggests that the establishment of theproteins of the outer membrane is one of the earlieststeps to take place in mitochondrial biogenesis.

DISCUSSION

The mitochondrial outer membrane is commonlycharacterized as a relatively simple phospholipid bi-

layer, broadly permeable to small proteins, ions, nu-trients, metabolic substrates, and other products ofmitochondria. Although broadly true, this simplifica-tion of the functions carried out by the mitochondrialouter membrane belies its involvement in complex,coordinated cellular processes such as apoptosis(Lindsay et al., 2011), organelle division (Kuroiwa,2010), mitochondrial inheritance (Koshiba et al.,2011a), lipid synthesis and trafficking (Osman et al.,2011), as well as selective interactions with the complexcytosolic environment in the processes ofmitochondrialprotein import and biogenesis (Walther and Rapaport,2009). The involvement of the mitochondrial outermembrane has often been incidental to the study ofthese processes, and the unequivocal assignment offunctions or protein components to the mitochondrialouter membrane is commonly avoided in the literature,significantly due to the difficulties in gathering suffi-cient evidence to make a convincing case for outermembrane localization. Studies such as the one pre-sented here have several important functions: confir-mation of some expected but unproven subcellularlocalizations; the identification of expected, but as yetuncharacterized, protein components of biological pro-

Figure 6. Transient expression of sev-eral fluorescently tagged fusion pro-teins causes apparent alterations inmitochondrial morphology/distribution.A, KDSB. Cells transformed with thisGFP-tagged protein consistently dis-played fewer but larger mitochondriathan was the observed norm for mito-chondrial outer membrane proteins.B, PECT-1. The mitochondria bunchtightly together at one pole of the cell.C, NADH:cytochrome B5 reductase.The mitochondrial population has anabnormal size distribution with manylarge mitochondria. D, Unknown pro-tein, commonly observed mitochon-drial morphology for comparison.






cesses; and as the basis for launching the investigationof novel, uncharacterized protein components, with thepotential to lead to the assignment of new and unex-pected biological functions. The analysis of the mito-chondrial outer membrane in plants is especiallyinteresting because the presence of the chloroplast inplant cells increases the complexity of the intracellularenvironment and possibly leads to the observed se-quence divergence across kingdoms (Macasev et al.,2000) in many of the protein components of the outermembrane involved in mitochondrial protein import(Lister et al., 2007). By systematically characterizing themitochondrial outer membrane proteome in plants, wehave identified proteins putatively involved in diverseprocesses, including signaling, cytoplasmic streaming,protein import, protein degradation, and membranebiosynthesis.

Developing a Proteomic Strategy for Characterizing theOuter Membrane Proteome

Developing a strategy for characterizing the pro-teome of one membrane from a multicompartmentedstructure within the cell is challenging due to the

multiple and overlapping potential sources of con-tamination and the probability that biochemical en-richment techniques will copurify multiple structuresat the same time. This is a recurring theme in subcel-lular proteomics, especially in subcellular membraneproteomics (Lilley and Dunkley, 2008). Combiningmethods that evaluate the protein content of enrichedmembranes relative to contaminating structures withvisual and biochemical assays such as in vitro andin vivo protein import provides a solid basis for de-termining subcellular and suborganelle localization.In combination, this is a highly complex and time-consuming process to undertake for a large range ofproteins. However, without it, many researchers aretoo often following up unexpected proteomic identi-fications by using genetic or functional assays, simplyto discover that the false discovery rate of the proteo-mics was too high in the first place.

Quantitative proteomic tools like spectral countingand iTRAQ offer powerful, unbiased analysis of theenrichment of known and unknown proteins betweensamples. However, we have found that the Exponen-tially Modified Protein Abundance Index and theNormalized Spectral Abundance Factor for quantita-

Figure 7. Coexpression of genes encoding outer mitochondrial membrane proteins. Publicly available microarrays analyzingwild-type tissues in the AtGenExpress developmental set (Schmid et al., 2005; E-AFMX-9) and during germination show thehighest expression of genes encoding inner membrane and outer membrane proteins during germination (Supplemental Fig. S5).The expression of genes encoding outer membrane proteins was analyzed during germination. Har, Seeds harvested on the day ofcollection from wild-type plants; 0 h, seeds 15 d after ripening, prior to imbibition; S, hours in cold stratification in the dark; SL,hours into continuous light after 48 h of stratification. Expression levels were made relative to maximum expression,hierarchically clustered (Euclidean distance and average linkage clustering), and five clusters were observed (C1–C5). A group ofgenes showing transient expression are indicated in the yellow box. Details for each of the tissues analyzed by microarrays areshown in Supplemental Table S6.

Duncan et al.





tive analysis of spectral counting (Ishihama et al., 2005;Zybailov et al., 2007) and parametric analysis of masstag abundances in iTRAQ spectra (Chong et al., 2006)are not immediately suited to assessments of enrich-ment. Typically, these processes were developed forthe analysis of small changes of protein abundance in abackdrop where most proteins are not changing inabundance. However, in enrichment studies (includ-ing the study presented here), most proteins arechanging in abundance, and significant enrichmentleads to the identification of low-abundance proteinsthat are not detected in more crude samples. Theseproblems affect the statistical analysis of both spectralcounting and iTRAQ data, as data sets are not nor-mally distributed in the cases of the most enriched andinteresting proteins from the perspective of subcellularlocation. Furthermore, while the degree of enrichmentmight be interesting in these types of studies, theprimary aim is to use quantification to give a qualita-tive assessment of cellular location. By using quanti-tative spectral counting and iTRAQ data to provide aqualitative assessment of location and coupling thisto independent confirmation data for this qualitativeassessment, we have developed a workflow for a con-fident assessment of the mitochondrial outer mem-brane proteome that is likely to be useful for researchon any specific membrane in the cell, particularly forthe analysis of membrane systems of low abundance(Fig. 1).There are three key aspects of this workflow. First,

selection of multiple independent prefractionatedsamples for comparisons with the enriched fractionsin order to independently cover likely causes of con-taminants; in on our case, we sought a control for othermitochondrial membranes (Mt IM), a control for thecoenrichment of contaminating structures in the Sucgradients (HSP-OM), and a control for nonmembraneproteins associating with the membrane during thecell fraction procedures (mitoplasts). Second, inclusionof proteins observed in enriched fractions and absentfrom prefractionated samples. Such proteins should beincluded despite issues related to the normality ofdistributions and limited spectral counts, as long asthe spectra are sufficient to prove identification, asthese include low-abundance proteins only foundthrough enrichment. Third, multiple confirmationstrategies that are independent of the enriched frac-tions are very helpful, especially those that relate totargeting of proteins in situ and/or in vitro to thelocation identified. In this study, we used a diverseliterature of counterclaims, GFP labeling in intact cells,and in vitro protein import. In addition to this, protein-protein interaction, in situ immunohistochemistry, andactivity assays could also be included for specificstudies (Millar et al., 2009).

Proteins of the Mitochondrial Outer Membrane Proteome

Using this workflow, 42 high-confidence identifica-tions of outer mitochondrial membrane proteins were

made (Table I). Only 16 of these proteins were previ-ously experimentally determined to be located in theouter mitochondrial membrane in plants (Werhahnet al., 2001; Chew et al., 2004; Lister et al., 2007;Arimura et al., 2008; Xu et al., 2008; Lee et al., 2009).These 16 proteins were largely made up of members ofthe outer mitochondrial membrane protein importapparatus (TOM complex and associated receptors)and the VDACs, specifically, the five known importreceptors (TOM 20-2, 20-3, 20-4, OM64, and Metaxin)and components of the central import pore (TOM 40,9-1, 9-2, 5, and 6) and VDAC1 to -4. Two other proteins,DGS-1 (At5g12290.1; Xu et al., 2008) and ELM1(At5g22350.1; Arimura et al., 2008), have also previ-ously been shown to be present in the outer mem-brane. Two proteins that were expected, but notdetected, in this study were TOM7 (Werhahn et al.,2001) and BIGYIN, the Arabidopsis ortholog toS. cerevisiae and human Fis1 (Scott et al., 2006).

In addition to the 16 known plant outer mitochon-drial membrane proteins, four other proteins were alsoinferred to be localized to the outer mitochondrialmembrane in plants, based on experimental evidencefrom other species (Fransson et al., 2003; Kozjaket al., 2003). Two of these proteins are the SAM50s(At3g11070.1 and At5g05520.1), which are evolution-arily conserved across kingdoms, and orthologs ofthese have also been reported to be mitochondrialouter membrane proteins in N. crassa (Schmitt et al.,2006) as well as S. cerevisiae (Zahedi et al., 2006).Similarly, the location of the two MIRO-like proteins,MIRO1 (At5g27540.1) and MIRO2 (At3g63150.1), werealso inferred to be located on the outer mitochondrialmembrane, based on orthology with the Gem1p genein S. cerevisiae (Frederick et al., 2004). Thus, experi-mentally confirming the localization of these in plants,to our knowledge for the first time, further supports avital and evolutionarily conserved role for the SAM50sand MIROs on the outer mitochondrial membrane.Evidence supporting this crucial role can be observedin Arabidopsis knockouts of MIRO1, which have beenshown to have a seed-lethal phenotype (Meinke et al.,2008; Yamaoka and Leaver, 2008), indicating that thisgene is essential for seed viability and normal plantdevelopment. In addition to the aforementioned pro-teins, a total of 27 proteins were reported as outermitochondrial membrane proteins in plants to ourknowledge for the first time in this study. Examinationof these revealed the presence of Hexokinase1 (HXK1;At4g29130.1), HXK2 (At2g19860.1), and Hexokinase-Like Protein1 (HXKL1; At1g50460.1) on the mito-chondrial outer membrane (Table I). The Arabidopsishexokinases have long been reported to be associatedwith mitochondria, first in 1983 (Dry et al., 1983;Damari-Weissler et al., 2007) and more recently withreference to the association of glycolytic enzymes withmitochondria (Graham et al., 2007). The presence of aputative transmembrane region of 20 to 25 aminoacids, high hydrophobicity at the N terminus (Sup-plemental Fig. S1, IV, VII, and XVII), the ability to






target GFP to mitochondria, combined with its enrich-ment in the outer membrane samples (Table I) and alack of detection of other glycolytic enzymes suggeststhat these proteins are authentic outer membraneproteins. The location of these hexokinases on theouter mitochondrial membrane in plants may be cru-cial for a role in the regulation of programmed celldeath, with recent findings in Nicotiana benthamianaimplying a role for mitochondrial hexokinases in thisprocess (Kim et al., 2006). Another study has alsoproposed a role for mitochondrial hexokinases ingenerating ADP to support oxidative phosphorylationandminimize the limitation of respiration by restraintson ATP synthesis, thereby playing a role in antioxidantdefense (da-Silva et al., 2004). Nevertheless, it appearsthat the presence of hexokinases on the outer mito-chondrial membrane positions these proteins ideallyfor a functional role in signaling.

Similarly, a NADH:cytochrome B5 reductase(At5g17770.1) might be expected to be in the outermembrane of mitochondria, based on the localizationof its S. cerevisiae ortholog (Haucke et al., 1997). Thereare two NADH:cytochrome B5 reductases in Arabi-dopsis; thus, the identification of this protein on theouter mitochondrial membrane distinguishes it fromother NADH:cytochrome B5 reductase activities inArabidopsis, such as the one encoded by At5g20080,which has been shown to bemitochondrial but is likelyto be located on the inner membrane (Heazlewoodet al., 2004). Notably, the protein shown to be on theouter mitochondrial membrane in this study; NADHcytochrome B5 reductase (At5g17770.1), has previ-ously been reported to be a microsomal enzymeassociated with the endoplasmic reticulum, eventhough no direct evidence was presented to deter-mine location (Fukuchi-Mizutani et al., 1999). A re-cent study has also shown that a mutation in thisNADH:cytochrome B5 reductase (At5g17770.1) sig-nificantly reduced the accumulation of hydroxyl fattyacids in developing seeds by 85%, confirming anessential role for this protein in lipid metabolism(Kumar et al., 2006). NADH:cytochrome B5 reduc-tases have been shown to catalyze the transfer ofelectrons from NADH to two molecules of membrane-bound cytochrome B5 (Strittmatter, 1965). Taken to-gether, these findings suggest that the reactionscatalyzed by both NADH:cytochrome B5 reductasesare likely to occur on the mitochondrial membranesand not on the endoplasmic reticulum, as assumedpreviously.

Novel Proteins of the Mitochondrial Outer Membrane

In addition to the proteins outlined above, a numberof other proteins were found to be located in the outermitochondrial membrane that could not have beenpredicted by sequence orthology alone. These proteinscan provide a molecular handle for processes thatoccur in mitochondria, where the identification ofmolecular components has been limited to date.

A Novel b-Barrel Protein

One of the proteins identified in the outer mem-brane proteome was a novel b-barrel protein(At3g27930.1), bringing the total number of distinctb-barrel proteins identified in the Arabidopsis mito-chondrial outer membrane to four (TOM40, SAM50,VDAC, and At3g27930.1; Table I). Amino acid se-quence alignments comparing this novel protein withother membrane b-barrel proteins (TOM40, SAM50,VDAC, MDM10, OMP85, and TOC75) from diversespecies revealed that this b-barrel protein is onlyfound in organisms with a green chloroplast, fromalgae to various plant species (data not shown). Al-though the function of this protein (At3g27930.1) isunknown (as annotated in TAIR 10), upon searchingthe literature, it was found that this gene has beenidentified as on the list of 437 proteins making up thepredicted Arabidopsis N-myristoylome (Boisson et al.,2003). Given that N-myristoylation is a permanentmodification that affects the membrane-binding prop-erties of cytoplasmic proteins, promoting their associ-ation with membranes, it was interesting to findAt3g27930.1 on this list. Taken together with the con-firmed outer mitochondrial membrane localization(Table I), high expression in seed and early germina-tion (Fig. 7), and the fact that this protein is plantspecific (Plant Specific Protein Database; Gutierrezet al., 2004), it can be speculated that this protein hasa crucial and plant-specific role in the interactionbetween cytoplasmic proteins and the outer mitochon-drial membrane, possibly in signaling or even as anovel import component.

Proteins Involved in the Synthesis ofMembrane Components

Three proteins that were confirmed to be located tothe outer mitochondrial membrane in the course ofthis study catalyze key steps in the synthesis of mem-brane components (At2g01460, At1g53000, andAt2g38670). At2g01460.1 contains two uridine mono-phosphate/cytidine monophosphate kinase-like do-mains and is predicted to catalyze the conversion ofuridine monophosphate/cytidine monophosphateto their diphosphate forms. It has previously beendemonstrated that a nucleoside diphosphate kinase(At4g11010.1) is present in the mitochondrial inter-membrane space (Sweetlove et al., 2001). Thus, thereaction of these two enzymes would yield cytidinetriphosphate from a cytidine monophosphate sub-strate with conversion of ATP to ADP. An additionalprotein found to be located to the outer membrane inthis study was PECT1 (At2g38670.1), which is a CTP:phosphorylethanolamine cytidyltransferase (Mizoiet al., 2006). This enzyme catalyzes the transfer ofphosphoethanolamine onto cytidine triphosphate +inorganic pyrophosphate to yield cytidine diphos-phoethanolamine (CDP-ethanolamine). The finalenzyme required for the production of phosphatidyl-

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ethanolamine is CDP-ethanolamine phosphotransfer-ase, the location of which is not yet characterized inArabidopsis but is found in the endoplasmic reticulumin S. cerevisiae (Jelsema and Morre, 1978). Interestingly,despite the fact that PECT1 is an intermediate in thispathway, it has been shown that when this gene isknocked out in Arabidopsis, a seed-lethal phenotypeis observed (Mizoi et al., 2006; Meinke et al., 2008).Thus, it appears that this role for PECT1 on the outermitochondrial membrane is essential for seed viabilityand development in Arabidopsis.In the course of confirming these proteins as bona

fide outer membrane proteins, full-length codingsequences were fused to the GFP coding sequenceand expressed using the 35S cauliflower mosaic viruspromoter, resulting in transient overexpression of theexperimental protein. Interestingly, in the case ofPECT1, this resulted in abnormally tight bunchingof the mitochondria at one pole of the cell as opposedto the regular evenly distributed morphology (Sup-plemental Fig. S1, IX), suggesting that PECT1 is notonly essential for catalyzing the reaction yieldingCDP-ethanolamine but that increasing the abundanceof this protein or perhaps the flux of its reaction canlead to abnormal phospholipid composition of theouter mitochondrial membrane or association of mi-tochondria. Also with a proposed role in membranesynthesis, At1g53000.1 encodes 3-deoxy-D-manno-2-octulosonic acid (Kdo) transferase, which catalyzesthe transfer of Kdo onto cytidine triphosphate to formCMP-Kdo (Seveno et al., 2010). This protein is orthol-ogous to the Escherichia coli protein KDSB, whichparticipates in the synthesis of KDO2-Lipid A, anessential component of the gram-negative bacterialcell wall (Opiyo et al., 2010). Basic local alignmentsearching showed that orthologous genes to each ofthe essential members of the bacterial KDO2-Lipid Abiosynthesis pathway are present in the Arabidopsisgenome and are predicted to be targeted to mito-chondria (Raetz and Whitfield, 2002; Heazlewoodet al., 2007). Taken together with the histochemicalevidence for the presence of Lipid A in eukaryoticorganisms (Armstrong et al., 2006), the evidencethat overexpression of this protein produced giantmitochondria (Fig. 4A) suggests the presence ofKDO2-Lipid A or a related compound in the outermembrane of mitochondria.

AAA-Type ATPases

Two related proteins containing ATPases associatedwith diverse cellular activities (AAA)-type ATPasedomains were identified on the outer membrane,At3g50930.1 and At3g50940.1, which are 67% identicalat the amino acid level and appear to be the result of arecent duplication in the Arabidopsis genome.Although the protein encoded by At3g50940 is

annotated as encoding a ubiquinol-cytochrome c re-ductase synthesis (BCS)-like protein, closer examina-tion reveals that it only contains the AAA-ATPase

domain in common and lacks the BCS domain. Inter-estingly, it differs from the AAA-ATPase proteasesthat are located on the inner membrane and displayscloser similarity to another AAA-ATPase, which hasbeen shown to be a calcium-binding protein whosefunction is still unclear but is dual localized to bothmitochondria and chloroplasts (Bussemer et al.,2009). Given that BCS1 was seen to be stably ex-pressed over normal development (Fig. 7) but hasbeen shown to be among the most stress-inducibletranscripts encoding mitochondrial proteins (VanAken et al., 2009), it may have a more specific rolein the repair of proteins following a wide variety ofstresses (Xu et al., 2011).

Proteins Involved in Signaling and Catabolic Processes

In addition to the stress-inducible AAA-type ATPa-ses discussed above, a number of other proteinsidentified in this study may have significant rolesin signaling. Hexokinases and MIRO have beenshown to have roles in signaling, with hexokinaseand hexokinase-like proteins shown to have roles inregulating reactive oxygen species levels and plantgrowth, respectively (Karve and Moore, 2009; Bolouri-Moghaddam et al., 2010), while MIRO proteins havebeen observed in regulating mitochondrial transportand morphology (Yamaoka and Leaver, 2008; Reiset al., 2009). Another protein that may be linked tosignaling is a TraB family protein (At1g05270.1), whichplays a role in the conjugative transfer of plasmids inbacteria (An and Clewell, 1994); however, its role ineukaryotic cells is still unknown. Nevertheless, theidentification of this protein implies a conserved rolefor it, both on bacterial membranes and evidently onthe mitochondrial outer membrane in plants. Anotherwell-conserved protein is Embryonic Factor1 (FAC1;At2g38280.1), for which the subcellular location haslong been discussed (Han et al., 2006), but in this studywe report an outer mitochondrial membrane localiza-tion for this protein, on the basis of both proteomic andGFP evidence. Interestingly, like PECT1 and MIRO1, ithas been observed that plants have a seed-lethalphenotype when FAC1 is knocked out (Xu et al.,2005; Meinke et al., 2008), indicating a vital role forthese outer mitochondrial membrane proteins inArabidopsis development.

CONCLUSION

While we have identified 42 proteins located at themitochondrial interface with the cell cytoplasm inplants, there are likely to be some membrane proteinsthat have not been identified in this study. Dual-targeted or multitargeted proteins would be excludedby the strategy undertaken, as they would be detec-ted in the contaminant fractions. The cell culturesystem used may not have components that may bepresent in photosynthetic cells, or they may be pre-sent at levels below the level of detection. No addi-






tional components of the SAM complex of the outermembrane were identified that might be expected tobe present, in comparison with the SAM complexfrom yeast. Nevertheless, 27 proteins were experi-mentally determined to be located on the outer mi-tochondrial membrane, to our knowledge for the firsttime in plants, and only one of these (At5g60730.1)has been consistently predicted to be mitochondrial(Heazlewood et al., 2007). Of the other 15 proteins,some had counterparts in S. cerevisiae or mammaliansystems and some have been previously shown to beassociated with the mitochondrial outer membrane.However, many can only be identified as on the outermitochondrial membrane based on direct experimen-tal analysis, as they are members of larger familiesthat have a variety of locations in the cell. Severalproteins that were identified had no functional anno-tations. For these, the discovery of outer membranelocalization provides, to our knowledge, the firstfunctionally related information for the proteinsencoded at these loci. Examination of the full list ofproteins identified (Table I) reveals that plant outermembrane functions exist beyond their well-estab-lished roles in protein and small molecule import.Importantly, we discovered that plant mitochondrialhomologs were not identified for a range of proteinsknown to be located in, and to function in, the outermembrane in other organisms. Examples include theB-cell lymphoma 2-like proteins, which play roles incell death in mammalian systems (Lindsay et al.,2011), the GTP protein b2 subunit, which plays a rolein regulating mitochondrial fusion in mammaliansystems (Zhang et al., 2010), Mim1, which plays arole in protein import in fungi (Stefan Dimmer andRapaport, 2010), and a peripheral-type benzodiaze-pine receptor that mediates cholesterol transport inmammalian cells but is located in the secretory path-way in Arabidopsis (Vanhee et al., 2011). Althoughwe were unable to conclusively rule out the existenceof these proteins on the mitochondrial outer mem-brane (expression of these proteins may be tissuespecific or of low abundance), it is likely that atleast some of the proteins that we identified withno known functions may fulfill similar roles. Thesefindings, together with the identification of fiveplant-specific genes (Plant Specific Protein Database;Gutierrez et al., 2004), including a novel b-barrelprotein (At3g27930.1) and a protein of unknownfunction (At5g55610.1), suggests that despite the con-served presence of mitochondria in eukaryotes, severalconstituents of the outer mitochondrial membranehave diverged and specialized in different species.

MATERIALS AND METHODS

Purification of Mitochondria from Arabidopsis

Suspension Cell Culture

Protoplasts were prepared from 6-d-old Arabidopsis (Arabidopsis thali-

ana) suspension cell cultures. Typically, 500 g fresh weight of cells was

collected by filtration and incubated for 3 h at 21�Cwith shaking at 30 rpm in

1 L of 0.4 M mannitol, 3.5 mM MES, pH 5.7, with 0.4% (w/v) cellulase and

0.05% (w/v) pectolyase (Yakult). Protoplasts were collected by centrifuga-

tion at 800g for 10 min, resuspended in 500 mL of 0.4 M Suc, 50 mM Tris, 3 mM

EDTA, 20 mM Cys, 0.1% (w/v) bovine serum albumin (BSA), pH 7.5, and

HCl and ruptured in a Potter-Elvehjem homogenizer. Cell debris was

removed by centrifugation at 2,500g for 5 min, and the organellar fraction

(supernatant) was pelleted at 20,000g for 20 min. A portion (1/16th) of this

material was removed and stored at 4�C for preparation of the HSP and

HSP-OM samples. The remaining organellar fraction was layered onto

discontinuous Percoll gradients consisting of 18%/25%/40% (v/v) Percoll

in 0.3 M mannitol, 10 mM TES, pH 7.5, and centrifuged at 40,000g for 40 min.

The 25%/40% (v/v) interphase was removed and added to 300 mL of 0.3 M

Suc, 10 mM TES, and 0.2% (w/v) BSA, pH 7.5 (Suc wash buffer), and

centrifuged at 20,000g for 15 min. Subsequently, the supernatant was

removed and the pellet was layered onto 40% (v/v) Percoll in Suc wash

buffer and centrifuged at 40,000g for 40 min. The gradient-purified mito-

chondria were removed and washed twice in Suc wash buffer. A total of 500

mg of this sample was removed and stored at 4�C for later analysis.

Typically, 100 to 150 mg of mitochondrial protein (Bradford assay) was

recovered.

The gradient-purified mitochondria were further purified by FFE based

on a method defined previously with several alterations (Eubel et al., 2007).

The gradient-purified mitochondria were first dispersed by passing them

back and forth between two 3-mL syringes through a 10-mm membrane

filter (Bio-Rad) and then passed through a 5-mm nylon syringe filter prior to

introduction to the FFE separation chamber. EDTA was excluded from the

FFE separation medium. FFE-purified mitochondria were collected by

centrifugation at 20,000g for 10 min and washed in Suc wash medium for

10 min.

Purification of Mitochondrial Outer Membranes andGeneration of Contaminant Samples

Outer membranes were purified according to a previously published

protocol (Werhahn et al., 2001). In addition to the mitochondrial outer

membranes, a “contaminant outer membrane” sample was generated by

treating the HSP collected from the protoplast disruption step in a similar

manner to the FFE-purified mitochondria. A second contaminant sample

primarily consisting of mitoplasts was taken from the 32%/60% (w/v)

interphase of the first Suc gradient. An inner membrane fraction was

prepared by diluting this in 11 mL of Suc wash medium before freezing/

thawing five times and pelleting at 504,000g for 1 h in a fixed-angle rotor. An

additional contaminant sample consisting of whole mitoplasts removed

from the same 32%/60% interphase but not subjected to the freezing/

thawing procedure was included in the iTRAQ experiment.

Western-Blot Analysis

Ten micrograms of membrane fractions and 20 mg of organelle fractions

per lane were separated by 14% (w/v) SDS-PAGE. Proteins were transferred

onto nitrocellulose membranes. Primary antibodies were as follows:

TOM40-1 (At3g20000.1) at 1:5,000 for 1 h at 21�C and developed according

to the manufacturer’s instructions (Roche; Carrie et al., 2009); KAT2 anti-

body (Germain et al., 2001) was used at 1:1,000 for 1 h at 21�C; Rubisco large

subunit and Calreticulin antibodies were obtained from Abcam and used at

1:500 for 18 h at 4�C; E1a antibody was obtained from Tom Elthon

(University of Nebraska) and used at 1:2,000 for 1 h at 21�C; COXII antibody

was obtained from Agrisera and used at 1:5,000 for 1 h at 21�C. Antibodies to

TOM20-3 (Lister et al., 2007), KDSB, SAM50, PECT1, and RISP were created

by purifying 63His-tagged recombinant proteins from Escherichia coli

expression using Profinia-based immobilized metal affinity chromatogra-

phy (Bio-Rad). The purified proteins were used to generate rabbit polyclo-

nal antibodies.

Proteinase K Titrations

A total of 250 mg of isolated mitochondria was resuspended in 0, 2, 8, 32,

and 64 mg mL21 proteinase K (Sigma-Aldrich) in 2.5 mL of 0.4 M Suc, 50 mM

Tris, 3 mM EDTA, 0.1% (w/v) BSA, pH 7.5, and HCl. Samples were incubated

Duncan et al.





on ice for 30 min before dilution in 30 mL of 0.4 M Suc, 50 mM Tris, 3 mM EDTA,

0.1% (w/v) BSA, pH 7.5, and HCl containing 1 mmol of Pefabloc SC (Roche).

Samples were centrifuged at 20,000g for 15 min, resuspended in Laemmli

sample buffer, and separated by SDS-PAGE.

MS

Label-Free Analysis and Statistical Assessment

Fifty micrograms of outer membrane, HSP-OM, and inner membrane

protein were individually digested overnight with trypsin (10:1), 10 mM

ammonium bicarbonate, and 20% (v/v) acetonitrile. Insoluble material was

removed by centrifugation at 20,000g for 5 min. Samples were then dried

down in a vacuum centrifuge and analyzed on an Agilent 6510 Q-TOF

mass spectrometer according to a modification of the methods reported by

Eubel et al. (2008), as outlined in detail in Supplemental Materials and

Methods S1. Spectra are available via the ProteomeCommons Tranche Project

hash: 3y16ynJWUCAiKGSfZNprN/paRI32nQA9XM0asqZItzPwnf2jEB/

zsnOAoSxno14kymUwnJbylKkk4cA2sGdLN52sAQ0AAAAAAAAHqg = =.

Peptide count data were compiled from three biological replicates each of

the outer membrane, HSP-OM, and mitochondrial inner membrane samples,

as outlined in detail in Supplemental Materials and Methods S1.

iTRAQ Labeling and Data and Statistical Analysis

A total of 100 mg of four samples (outer membrane [Mt OM], contaminant

outer membrane [HSP-OM], mitoplast, and inner membrane [IM]), each

created by pooling three biological replicates, were analyzed according to a

modification of the method reported previously (Shingaki-Wells et al., 2011) as

outlined in Supplemental Materials andMethods S1. Quantitation was carried

out using an in-house quantitation method that carried out protein identifi-

cations and quantitation on isobaric tags of mass 114 to 117 at the peptide

level. Ratios for individual peptide matches were obtained from peptides

meeting the minimum criteria outlined above and were then combined to

determine ratios for protein hits using a weighted average. The in-house

method of identification and quantitation, outlier removal, minimum number

of required peptides, and definitions of statistical confidence internals are

outlined in detail in Supplemental Materials and Methods S1.

In Vitro Imports

In vitro import of putative outer membrane proteins into isolated mito-

chondria was performed as described previously (Lister et al., 2007). Data are

provided in Supplemental Figure S3, II, III, and IV.

GFP Analysis

Full-length coding sequences (with the exception of At4g17140.2) of

putative outer membrane proteins were cloned into the Gateway cassette

cloning system according to the manufacturer’s instructions (Invitrogen;

http://invitrogen.com/). Coding sequences were transferred into GFP vec-

tors and cotransformed into Arabidopsis suspension cell culture with a red

fluorescent protein control by particle bombardment as outlined previously

(Carrie et al., 2009). Data are provided in Supplemental Figures S1 to S4.

Arabidopsis Microarray Analysis

The Arabidopsis AtGenExpress developmental data set was downloaded

as CEL files (E-AFMX-9) from the MIAME ArrayExpress database (http://

www.ebi.ac.uk/arrayexpress/). These CEL files, in addition to the 30 CEL

files that analyzed expression during germination, were imported and quan-

tile normalized together to enable comparability across these arrays using

Partek Genomics Suite version 6.5. Details for each tissue are shown in

Supplemental Table S5. Once normalized, expression values were made

relative to maximum expression and hierarchically clustered on Euclidean

distance using average linkage clustering. Note that four genes, At1g24267,

At3g11070, At3g50940, and At5g22350, were not represented on the ATH1

microarray. This method of normalization and clustering has been used

effectively before to examine coexpression (Narsai et al., 2010).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under the accession numbers listed in Supplemental Table S8.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Members of the mitochondrial outer membrane

proteome, as shown by fluorescence microscopy of transiently trans-

formed Arabidopsis cell culture or onion (Allium cepa) cells with full-

length coding sequences fused to GFP.

Supplemental Figure S2. Members of the mitochondrial outer membrane

proteome that are also found in other subcellular locations, as shown by

fluorescence microscopy of transiently transformed Arabidopsis cell

culture or onion cells with full-length coding sequences fused to GFP.

Supplemental Figure S3. Members of the putative mitochondrial outer

membrane proteome that have been shown to be mitochondrial but not

outer membrane by fluorescence microscopy of transiently transformed

Arabidopsis cell culture or onion cells with full-length coding sequences

fused to GFP.

Supplemental Figure S4. Members of the putative mitochondrial outer

membrane proteome that have been shown not to be mitochondrial by

fluorescence microscopy of transiently transformed Arabidopsis cell

culture or onion cells with full-length coding sequences fused to GFP.

Supplemental Figure S5. Coexpression of genes encoding outer mito-

chondrial membrane proteins.

Supplemental Table S1. List of the 185 proteins detected in two or more of

the outer membrane-enriched biological replicates (www.Arabidopsis.

org).

Supplemental Table S2. List of the 64 members of the putative outer

membrane proteome with evidence for identification and enrichment in

the outer membrane fraction (www.Arabidopsis.org).

Supplemental Table S3. Summary of the location evidence gathered in the

course of this research.

Supplemental Table S4. Summary of data gathered from iTRAQ analysis

of the outer membrane-enriched and three prefractionation samples.

Supplemental Table S5. Details of the germination microarrays and

developmental tissue set (Schmid et al., 2005; E-AFMX-9).

Supplemental Table S6. Details of the genes defined as encoding inner

mitochondrial membrane proteins.

Supplemental Table S7. Details of polypeptide identifications resulting

from the in-gel digestion of bands excised from the gel pictured in

Figure 3A

Supplemental Table S8. Sequence data.

Supplemental Materials and Methods S1. Detailed MS methods.

Received July 9, 2011; accepted August 31, 2011; published September 6, 2011.

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