Analysis of the is Mitochondrial Proteome

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Analysis of the Arabidopsis Mitochondrial Proteome1

A. Harvey Millar*, Lee J. Sweetlove, Philippe Giege, and Christopher J. Leaver

Department of Biochemistry, Faculty of Medicine and Dentistry, and the Plant Sciences Group, Faculty ofAgriculture, The University of Western Australia, Crawley 6009, Western Australia, Australia (A.H.M.); andDepartment of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom(L.J.S., P.G., C.J.L.)

The complete set of nuclear genes that encode proteins targeted to mitochondria in plants is currently undefined and thusthe full range of mitochondrial functions in plants is unknown. Analysis of two-dimensional gel separations of Arabidopsiscell culture mitochondrial protein revealed approximately 100 abundant proteins and 250 low-abundance proteins. Com-parison of subfractions of mitochondrial protein on two-dimensional gels provided information on the soluble, membrane,or integral membrane locations of this protein set. A total of 170 protein spots were excised, trypsin-digested, and

matrix-assisted laser desorption ionization/time of flight mass spectrometry spectra obtained. Using this dataset, 91 of theproteins were identified by searching translated Arabidopsis genomic databases. Of this set, 81 have defined functions basedon sequence comparison. These functions include respiratory electron transport, tricarboxylic acid cycle metabolism, aminoacid metabolism, protein import, processing, and assembly, transcription, membrane transport, and antioxidant defense. Atotal of 10 spectra were matched to Arabidopsis putative open reading frames for which no specific function has beendetermined. A total of 64 spectra did not match to an identified open reading frame. Analysis of full-length putative proteinsequences using bioinformatic tools to predict subcellular targeting (TargetP, Psort, and MitoProt) revealed significantvariation in predictions, and also a lack of mitochondrial targeting prediction for several characterized mitochondrialproteins.

The mitochondrion is the organelle within the eu-karyotic cell that is primarily concerned with thesynthesis of ATP in the fundamental process known

as respiration. The origins of this organelle can betraced back to an event in which a prokaryotic cellwas engulfed by another prokaryote to form a celllineage containing two independent genomes. Overtime, these genomes became codependent and mito-chondria lost the ability to be viable organisms out-side the host cell. A substantial transfer of geneticinformation occurred from the mitochondrial to thenuclear genome during this time. Today, it is pre-dicted that mitochondria synthesize 2% to 5% of theproteins required for their function, with the remain-ing 95% to 98% of proteins required encoded by thenuclear genome and targeted back to the mitochon-

dria as protein precursors using encrypted targetinginformation in the protein sequence (Gray et al.,1999). The detection of these encryptions and thusidentification of the full set of these genes within thenuclear genome is a major challenge for biologists.

The recent sequencing of whole genomes has en-couraged an increasing effort to develop bioinfor-matic tools to predict the cellular localization of pu-

tative protein sequences. Psort was developed as anexpert system that uses a set of 100 if-then-type ofrules based on analysis of characterized protein se-quences from a variety of subcellular locations (Na-kai and Kanehisa, 1992). MitoProt was developed topredict mitochondrial targeting and presequencecleavage sites based on a set of 47 known character-istics of presequences and cleavage sites (Claros andVincens, 1996). More recently, TargetP, based on neu-ral network programming, was developed to predicttargeting of protein sequences to chloroplasts, mito-chondria, and the secretory system using a knowl-edge based derived from Swiss-Prot sequence entries

(Emanuelsson et al., 2000).The full sequences of the five chromosomes from

the model plant Arabidopsis were recently published(The Arabidopsis Genome Initiative, 2000). The anal-ysis of this data included a full genome analysisusing TargetP that predicted the number of nuclear-encoded, mitochondrial-targeted gene products. Thisnumber could be as high as 2,897, or, using a 0.95specificity cut-off in TargetP, it could be as low at349. The breadth of this prediction range (8.1-fold)was in stark contrast to the range for chloroplasttargeting (0.7-fold) and secretory pathway targeting(0.07-fold) when using the same high- and low-

stringency settings in TargetP.

1 A.H.M. was supported by an Australian Research CouncilAustralian Postdoctoral Fellowship. This work was also supported

by the Biotechnology and Biological Sciences Research Council (toC.J.L.) and by the University of Western Australia Small GrantsScheme (to A.H.M.).

* Corresponding author; e-mail [email protected];fax 618 9380 1148.

Article, publication date, and citation information can be found

at www.plantphysiol.org/cgi/doi/10.1104/pp.010387.

Plant Physiology, December 2001, Vol. 127, pp. 17111727, www.plantphysiol.org 2001 American Society of Plant Biologists 1711


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Analysis of the presequences that direct proteinsto mitochondria in plants shows substantial differ-ences to the consensus sequences present in yeast(Saccharomyces cerevisiae) and mammals. This meansthat identifications in plants cannot simply rely on

existing bioinformatic tools (Sjoling and Glaser,1998). In addition, a range of proteins that areknown to be targeted to plant mitochondria do notutilize cleavable N-terminal presequences and thusremain undetected by algorithms or training regimes

based on identification of these signal peptides. Ex-amples include a range of outer membrane proteinsand also the inner membrane superfamily of carrierproteins (Laloi, 1999). All the prediction programsavailable have been largely established and/ortrained on non-plant sequences, and thus the robust-ness of these tools for prediction in plants is stilluncertain.

Density gradient purification of plant mitochon-dria has been optimized from a range of plant tissuesand allows the rapid and high-purity recovery ofthese organelles (Day et al., 1985; Neuberger, 1985).Biochemical investigations have revealed that iso-lated plant mitochondria share many similarities tothose from animals and fungi. However, plant mito-chondria also contain additional features such asnon-phosphorylating bypasses of the electron trans-port chain, specialized metabolite carriers, and en-zymes involved in the synthesis of folate, lipoic acid,and vitamin C (Rebeille et al., 1997; Bartoli et al.,2000; Gueguen et al., 2000). Only a few of the nuclear

genes encoding proteins that maintain these uniquefunctions have been identified. In addition, the pre-dictions by TargetP suggest that an array of otherproteins whose functions have yet to be character-ized are present in the nuclear genome of plants.

Several analyses of subcellular proteomes fromArabidopsis have been reported for the plasmamembrane and endoplasmic reticulum systems(Santoni et al., 1998; Prime et al., 2000) and thechloroplast (Peltier et al., 2000). These studies haveused a combination of Edman degradation and massspectrometry (MS)/MS-based sequencing to identifyproteins from two-dimensional gels. To determine

which putative proteins from the Arabidopsis ge-nome sequence represent constitutively expressedproteins for mitochondrial function, we have usedtwo-dimensional analysis of purified Arabidopsismitochondria coupled with peptide mass fingerprint-ing identification. This approach aims to identifyproteins localized in plant mitochondria, thus pro-viding new insights into the function of plant mito-chondria and also providing a larger set of experi-mentally characterized mitochondrial proteins. Thisset of experimental knowledge could be used to im-prove neural network and/or rule based program-ming for targeting prediction of products from plant

nuclear genomes.

RESULTS

Isolation of High-Purity Mitochondria fromArabidopsis Cell Culture

The integrity of a subcellular proteome, such as

that of mitochondria, is largely dependent on thepurification of the isolated compartment away fromother cellular contaminants. We have used two Per-coll gradient density separations that yield mitochon-dria that are essentially free of contamination bycytosol, peroxisomes, plastids, and other membranes(Fig. 1). On the first step-gradient, mitochondria

band at the interface of the 23% and 40% Percoll steps

Figure 1. Isolation of Arabidopsis mitochondria by density centrifu-gation. The organelle pellet from homogenized Arabidopsis cellculture was loaded onto a Percoll step gradient consisting of steps of40% (fractions 3135), 23% (fractions 1130), and 18% Percoll(fractions 110; A). After centrifugation, mitochondria were recov-ered from the 40%:23% interface (fractions 2635) and were loadedonto a self-forming Percoll gradient containing 28% Percoll (B).One-milliliter fractions were collected from both gradients (from topto bottom) and the activities of cytochrome c oxidase (f), catalase(E), alkaline pyrophosphatase (), and alcohol dehydrogenase ()were assayed in each fraction. Values are expressed as a percentage

of the total activity in the initial cell extract.

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(corresponding to the peak of cytochrome c oxidaseactivity in fractions 2635; Fig. 1A). These mitochon-dria were contaminated by peroxisomes and plastidsas revealed by the activity of catalase and alkalinepyrophosphatase, respectively. However, alcohol de-

hydrogenase could not be detected in these fractions,indicating the absence of contamination by cytosol.Interestingly, plastids resolved into three separatepopulations, and the presence of carotenoids (a com-monly used marker for plastids) only correlated withone of the plastid peaks (fractions 1320; data notshown). Thus, for Arabidopsis cells, the presence ofcarotenoids is not a reliable marker for the presenceof all types of plastids. To further purify the mito-chondria, a second self-forming gradient consistingof 28% Percoll was employed (Fig. 1B). Mitochondriaformed a broad band in the upper part of the gradi-ent, corresponding to a peak of cytochrome c oxidaseactivity at fraction 5. The mitochondrial band was

free of contamination by peroxisomes (catalase activ-ity was not coincident with cytochrome c oxidaseactivity, instead forming a peak lower down the gra-dient between fractions 2329). A small peak of alka-line pyrophosphatase activity did coincide with themitochondrial peak, indicating a slight contamina-tion by plastids. However, based on the activity ofthe marker enzymes in fractions 1 through 14 incomparison with that present in the initial cell ex-tract, the yield of mitochondria was 12.4%, whereasthe yield of plastids in the same fractions was only0.2%. This level of contamination is sufficiently mi-nor that it is unlikely that we would detect plastid

proteins in two-dimensional gels of the mitochon-drial fraction.

Integrity and Function of IsolatedArabidopsis Mitochondria

We assayed the integrity and function of the puri-fied mitochondria to ensure that proteins were not

being lost by rupture during isolation and that keyfunctions were maintained. The outer membrane ofmitochondrial samples was found to be 97% intact

based on the latency of cytochrome c oxidase activityinitiated by exogenously added cytochrome c (Table

I). Whole electron transport chain activities showed

that oxygen consumption by the mitochondria couldbe supported by succinate, external NADH, or a combination of malate and pyruvate. Oxygen consump-tion supported by each of these substrates was sig-nificantly and transiently stimulated by addition of

ADP. These data suggested the presence and func-tion of the electron transport chain, the F1F0-ATP

synthase, the tricarboxylic acid (TCA) cycle in themitochondrial matrix, and the integrity of the innermembrane. Oxygen consumption by mitochondrialsamples was largely inhibited by addition of thecytochrome oxidase inhibitor, KCN, and the smallremaining rate was inhibited by n-propylgallate(nPG), an alternative oxidase inhibitor (Table I).

Fractionation of Mitochondrial Proteins

Mitochondrial protein samples were further frac-tionated based on the degree of membrane associa-

tion (DMA). Total protein (A), soluble protein (B),membrane protein (C), and integral membrane pro-tein (D) samples were separated by two-dimensionalgel electrophoresis (Fig. 2, AD). This analysis re-vealed varying patterns of protein spots in each gelcorresponding to the relative abundance of each pro-tein in the different compartments. Based on thesegels, an overlay of the whole mitochondrial proteingel was made with protein spots highlighted thatcould be clearly identified by spot abundancechanges to be peaking in amount in a particularfraction. In total, 163 protein spots were tracked forlocation in this manner. A set of 43 protein spots

were identified as soluble proteins (S; present in A,peaked in B, and largely absent in C and D). A totalof 21 protein spots were identified as peripheralmembrane proteins (P; present in A, absent in B,peaking in abundance in C, but absent in D). Only 18protein spots were deemed integral membrane (I;present in A, absent in B, and present in C and D insimilar abundance). An additional 81 spots could not

be easily assigned to any group as they appeared inabundance in several fractions (circled, unmarked).Allocation of protein spots to DMA groupings was

based on densitometry analysis of gels by ImageMas-ter two-dimensional analysis software (Amersham

Pharmacia Biotech, Sydney, Australia). Allocations to

Table I. Oxygen consumption by isolated Arabidopsis mitochondria following addition of different substrates, effectors, and inhibitors

Respiratory assays were performed according to Materials and Methods. Means SD (n 3) for oxygen consumption rates are presentedand outer membrane integrity is presented as a percentage of intact mitochondria.

Substrate ADP ADP KCN nPG

nmol O2

min1 mg1 protein

NADH 233 32 123 13 10 5 2 2Succinate 250 34 122 19 15 5 1 2NADH and succinate 390 52 166 24 25 7 1 2Malate and pyruvate 172 18 66 11 7 3 1 1Cytochrome c 640 86 3 2

Outer membrane Integrity (%) 97

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Arabidopsis Mitochondrial Proteome

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Figure 2. Two-dimensional separations of Arabidopsis mitochondrial proteins (pI 310) fractionated on the degree ofmembrane association. A, Total mitochondrial proteins; B, soluble proteins; C, total membrane proteins; D, integralmembrane proteins based on Na2CO3 treatment; and E, predicted localization of 165 protein spots on the basis ofdistribution in A through D. Circled, black spots are annotated as I, S, or P. Circled, unannotated gray spots could not beassigned as I, S, or P based on their distribution between the gels. Numbers on the xaxis are pI and numbers on the yaxisare apparent molecular mass (kilodaltons) in E. A through D, Ticks marks show the position of pI 3 to 10 and the sixmolecular mass markers presented in E.

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Ta

ble

II.

(Continuedfrom

previouspage.)

Sample

DB

Entry

De

tailsofMatch

No.MP

Percentage

Covered

MM(Da)

Match

pl

Match

MolecularMass

(Da)Gel

plGel

DMA

TargetP

MitoProt

Psort

GRAVY

23

TE

O24345

ATPsynthase,

subunit

10

36

49,135

5.3

57,000

5.4

PM

164

TE

Q9SJ12

ATPsynthaseFadsubunit

8

38

27,597

6.6

31,000

5.3

PM

M1

0.9000

N

(0.760)

0.656

1

GB

T46100

ATPsynthaseDsubunit

8

48

19,586

5.1

23,000

4.8

PM

3

0.5303

C

(0.450)

0.896

170

TE

Q9SUU5

Ubiquinol-cytochromec

reductase

7

40

14,527

9.9

15,000

9.9

N

M4

0.8264

C

(0.450)

0.633

151

TE

Q9SSB8

Cytochro

mecoxidaseVB

subunit

6

29

18,583

5.7

15,000

4

N

C4

0.9175

Mma(0.759)

0.321

82

TE

Q9SRR8

Adrenodoxin/mitochondrial

ferrido

xin

5

30

17,602

8.3

17,000

5.7

N

C5

0.9430

C

ls(0.623)

0.275

10

TE

Q9SRK3

MNsupe

roxidedismutase

8

55

25,444

8.7

26,000

6.5

S

M2

0.9609

Mma(0.699)

0.338

79

GB

BAA96953

UQbiosynthesismethyl-

transfe

rase

5

25

32,290

6.7

31,000

5.9

N

M2

0.9970

Mma(0.920)

0.166

Membranecarriers

57,96

TE

Q9SRH5

V-DEPanion-selective

channelprotein

8,8

45,47

29,425

9

30,00031,000

9.39.5

IM

2

0.1328

N

(0.760)

0.114

14,128

TE

Q9SMX3

V-DEPanion-selective

channelprotein

7,8

43,51

29,211

8.4

30,000

8.19.2

IM

3

0.1576

P(0.560)

0.183

RN

Ametabolismandtranslationalapparatus

47,94,168

TE

Q9ZT91

Mitochondrialelongation

factorTU

12,12,22

41,39,5

2

49,410

6.6

43,000

5.4

6.2

N

C5

0.8743

C

ls(0.570)

0.117

102,103

TE

Q9SHD6

Mitochondrialelongation

factorG

9,20

16,31

83,112

6.3

86,000

6.7

6.8

S

M4

0.8737

ER

m(0.600)

0.259

145

TE

O82505

Translationelongationfac-

torTS

10

27

43,566

6

40,000

5.3

N

M4

0.9538

Mma(0.704)

0.129

2

TE

Q39105

Gly-rich

RNA-bindingpro-

tein

4

41

15,702

6.7

15,000

5.1

S

M4

0.7432

P(0.640)

0.294

163

GB

BAB01770

Dead-boxRNAhelicase

17

32

65,359

9.5

61,000

9.3

N

M3

0.9822

Mma(0.867)

0.315

He

atshockprotein,chaperonin,andproteinproc

essing

97

GB

116229

Chaperonin60

4

11

61,351

5.7

66,000

5.2

S

M3

0.9898

Mma(0.685)

0.069

45

TE

O49314

Chaperonin60

11

35

55,254

5.3

66,000

5.3

S

1

0.0495

C

(0.450)

0.023

32,99

TE

Q9SZJ3

Heatsho

ckprotein70

10

18,22

71,174

5.3

67,000,74,000

4.8,5.1

S

C5

0.8846

C

ls(0.520)

0.298

28

GB

T49939

Heatsho

ckprotein70

13

35

72,991

5.6

74,000

5.3

S

M4

0.9879

Mma(0.907)

0.306

30

TE

Q9S7E7

Putative

heatshock

protein

90

18

33

90,956

5.3

94,000

4.8

IM

M4

0.9919

Mma(0.793)

0.600

31

TE

Q9SIF2

Putative

heatshock

protein

90

9

16

88,663

5

90,000

4.5

N

C3

0.7324

N

(0.600)

0.552

65

GB

BAB09831

ATP-DEP

protease(clpP-

like)

8

30

26,283

6.7

27,000

6.4

S

M3

0.7529

P(0.640)

0.031

25,167

TE

Q9SGA7

Processin

gpeptidase

sub-

unit

12,18

34,39

59,160

6.7

56,000,61,000

5.6,6.2

IM

M2

0.9910

Mma(0.920)

0.342

17

TE

Q9ZU25

Processin

gpeptidase

sub-

unit

12

32

54,402

6.2

54,000

5.3

IM

M3

0.9556

Mma(0.898)

0.126

13

TE

O04331

Prohibitin

9

51

30,400

7.7

31,000

7.5

IM

S2

0.5098

C

(0.450)

0.013

(Tablecontinuesonfollowingpage.)

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S, P, and I were only made if a protein spot abun-dance was more than 5-fold in the present versusabsent gels for each class. For example, the abun-dance of an S protein in A and B was more than5-fold that in C and D. The S, P, and I classes and no

class (N) are also annotated on Table II in the DMAcolumn.

MALDI-ToF Peptide Mass Fingerprinting ofArabidopsis Mitochondrial Proteome

A set of 170 protein spots, including those identi-fied in Figure 2 and several other less-abundant pro-teins, were chosen because they were reproduciblyobserved in the mitochondrial proteome throughoutthe different experiments undertaken. These protein

spots were excised from whole mitochondria profilegels and were in-gel digested for further MS analysisof the resulting peptide fragments (Fig. 3). MALDI-ToF provided a peptide mass spectrum for each pro-tein spot. From this total of 170 proteins, 15 samples

provided poor spectra that could not be used forfurther analysis (6, 29, 36, 42, 52, 63, 64, 70, 71, 75, 113,114, 123, 134, and 135), and the remaining 155 wereused for database (DB) searches to identify genesencoding proteins with similar peptide mass finger-prints. A total of 81 of the spectra were matched to 68predicted Arabidopsis protein sequences in the DBwith known function based on functional studies orsequence comparison with proteins of known func-tion (Table II). A further 10 spectra matched to ninepredicted Arabidopsis protein sequences, largely

Figure 3. Two-dimensional separation of Arabidopsis total mitochondrial proteins (pI 310). A total of 170 protein spotsfrom this map were excised, digested, and analyzed by MALDI-ToF to yield a peptide mass fingerprint for DB searching.Numbers indicate spot number for comparison with text and Table II. Numbers on the x axis are pI and numbers on the y

axis are apparent molecular mass (kilodaltons).

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from genome sequencing for which no function hasbeen identified by functional studies or comparativegenomics. The apparent and predicted molecularmass and pI of the matched samples are consistentwith expectation. In cases where a mitochondrial

targeting presequence is present, we expect a de-crease of 1 to 4 kD between predicted and apparentmolecular mass, and we expect a more major shift inthe pI of the protein in the acidic direction followingthe removal of the typically basic presequence. Incases where no presequence is present (such as theporins, TOM40, and aconitases), the apparent andpredicted molecular mass and pI values are muchcloser. Overall, the sequences, excluding thoseknown not to contain presequences, apparent molec-ular masses were 1.5 0.5 kD smaller than precursorproteins predicted masses. The pI values of proteinspots observed on gels were also shifted 0.85 0.15units in the acidic direction compared with predicted

precursor protein pI values (Table II).

Membrane Channels/Carriers

The voltage-dependent anion-selective channelproteins, or porins, allow channel transport of smallmolecules (8 kD) across the outer mitochondrialmembrane. Four proteins in the Arabidopsis mito-chondrial profile (14, 57, 96, and 128) were matchedto two separate Arabidopsis genes encoding thisclass of channel protein. All four of these proteinswere identified in the integral membrane protein set

based on the DMA analysis of Figure 2.

Protein Import, Processing,Chaperonins, and Degradation

Three types of the classical heat shock/chaperoninproteins were identified, those in the 60-, 70-, and90-kD classes (45, 97, 32, 99, 28, 30, and 31). Thepartner protein of HSP60, known as HSP10, was notidentified; it would have been too small (approxi-mately 10 kD) to be observed on the SDS-PAGE gelsin these experiments. The new class of molecularchaperonins recently characterized in mammalianmitochondria, known as prohibitins (Nijtmans et al.,2000), were also identified in our mitochondrial sam-ples. A total of five prohibitin-like proteins wereidentified (13, 58, 156, 166, and 169) and matched asthe products of five separate genes in Arabidopsis. Inaddition, the TOM40 receptor for protein import onthe outer mitochondrial membrane was identified(98), along with both subunits of the mitochondrial-processing peptidase responsible for removal ofN-terminal targeting presequences from mitochon-drial proteins (17, 25, and 168). A clpP-like ATP-dependent protease probably involved in proteolysisin mitochondria (65) was also identified. Recently,antibodies raised to a recombinant Arabidopsis pro-

tein, designated by homology to be a putative plant

mitochondrial clpP, have been used to localize across-reacting protein in mitochondria from pea (Pi-sum sativum; Halperin et al., 2001). In yeast, clpP-likeproteases are known to be associated in large super-complexes with prohibitins in mitochondria, and ge-

netic evidence suggests prohibitins have a negativeregulatory effect on protease function (Steglich et al.,1999). Further investigation of a prohibitin/ATP-dependent protease complex in plant mitochondria iswarranted.

TCA Cycle Enzymes

Proteins from all of the TCA cycle enzyme com-plexes were identified, and these included subunitsof pyruvate dehydrogenase complex (44, 132, 18, 15,108, and 86), citrate synthase (104 and 33), isocitratedehydrogenase (112), 2-oxoglutarate dehydrogenase

(15, 108, and 86), succinyl coenzyme A (CoA) ligase(3 and 100), succinate dehydrogenase (46), fumarase(87), and malate dehydrogenase (56 and 34). The twopyruvate dehydrogenase E1 subunits (44 and 132)share 87% sequence identity and are only 50% to 52%identical to the plastidic E1-subunit sequence (GBU80185), suggesting they are mitochondrial E1 sub-units. Notable absences from the list include NAD-malic enzyme that catalyzes the conversion of malateto pyruvate in the plant TCA cycle. The activity ofthis enzyme was present in Arabidopsis mitochon-dria (data not shown). Previous studies by several ofthe authors in potato mitochondria showed that the- and -subunits of NAD-malic enzyme enter two-dimensional gels and cross-react with antibodiesraised to NAD-malic enzyme (Jenner et al., 2001).None of the E2 subunits of pyruvate or 2-oxoglu-tarate dehydrogenase complexes were identified, al-though these proteins are present in two-dimensionalgels in mitochondrial samples from other plant spe-cies (data not shown). The modification of these pro-teins by lipoic acid moieties might explain why theyare not identified by mass fingerprinting, as thesemodifications will alter the apparent molecularmasses of peptides derived from these proteinsequences.

Aconitase

In yeast, a single nuclear gene encodes aconitase,and inefficient import of this protein is believed toexplain the cytosolic and mitochondrial localizationof this protein (Gangloff et al., 1990). It had beensuggested that a single gene also encoded aconitasein Arabidopsis (Peyret et al., 1995); however, subse-quent genome sequencing has shown there are fouraconitase genes in this plant species. We have iden-tified four aconitase proteins (101, 116, 165, and 21) asthe products of two aconitase genes (Q9SZ36 andQ9SIB9) in the mitochondrial profile. The predicted

protein sequences from these genes are only 75% and




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84% identical to the original aconitase cloned andentered into SWISS-PROT (Q42560) as the cytoplas-mic aconitase hydratase by Peyret et al. (1995). Nei-ther of the two predicted protein products has puta-tive mitochondrial targeting sequences. The

mechanism of import of these large proteins into themitochondrial matrix in the absence of a presequenceis not known.

Lipoamide Dehydrogenase

Two distinct lipoamide dehydrogenase proteinswere identified as the products of Q9M5K2 andAAF34795. These predicted proteins are identical inlength (507 amino acids) and share 94% identity atthe amino acid level. The power of peptide massfingerprinting is shown by the ease of differentiationof these two proteins by peptide matches. Only four

peptides in both spectra of identical size werematched to identical sequence in both predicted pro-tein sequences, an additional six to eight peptides ineach case matched to regions with one or two aminoacid substitutions altering peptides mass in each pre-dicted protein sequence (data not shown). The pres-ence of these two mitochondrial proteins is in con-trast to a long-standing debate in the literature aboutthe number of lipoamide genes and protein productsthat serve as the dehydrogenases for the 2-oxo acidmultienzyme complexes and Gly decarboxylase com-plex in mitochondria and chloroplasts. It has beenheld that a single product of a single gene encodinglipoamide dehydrogenase was targeted to both or-ganelles and partnered all these multienzyme com-plexes. Recently, two genes encoding plastid specificlipoamide dehydrogenases have been identified(Lutziger and Oliver, 2000). The same authors clonedand entered both of these mitochondrial lipoamidedehydrogenases sequences (Q9M5K2 and AAF34795)into public DBs in 2000. This is the first evidence toour knowledge that these two genes are translated toform lipoamide dehydrogenase proteins that accu-mulate in plant mitochondria. Further, it appearsthere is significantly more Q9M5K2 than AAF34795protein in our mitochondrial samples (Fig. 3).

Electron Transport Chain

Representative subunits from each of the classicalelectron transport chain complexes were identified.These included five subunits of complex 1 (NADH-ubiquinone [UQ] oxidoreductase 72, 80 20, 22, and91), three subunits of complex III (UQ-cytc oxi-doreductase 171, 17, 168, and 25), five subunits ofcomplex V (ATP synthetase complex, 1, 4, 24, 23, and164), and one subunit each from complex II (succi-nate dehydrogenase, 46) and complex IV (cyto-chrome c oxidase, 151). No sequence entry for the-subunit of the mitochondrial ATP synthase from

Arabidopsis could be found by the authors. Matches

to the spectrum from spot 23 were found to the-subunit from a wide range of other plant species; inTable II, the -subunit from Sorghum bicolor wasmatched to 205. The reason for the absence of anentry for this major mitochondrial protein from Na-

tional Center for Biotechnology Information, Trans-lated European Molecular Biology Laboratory Nucle-otide Sequence Database, and the The ArabidopsisInformation Resource DBs is unclear. No peptidefingerprint spectra matched to protein sequences forthe non-phosphorylating bypasses of the plant elec-tron transport chains, notably the alternative oxidaseprotein sequences (Saisho et al., 1997) or the recentlyidentifiedputativerotonone-insensitiveNADH-dehy-drogenase protein sequences (Rasmusson et al.,1999). This absence was despite the evidence fromTable I of the operation of both of these bypasses inintact Arabidopsis mitochondria. A protein with sig-nificant homology with the mitochondrial class of

ferrodoxins in mammals known as adrenodoxins wasalso found (82). In mammals, this class of ferrodoxinspecifically function in a redox chain with an adreno-doxin reductase to reduce cytochrome P450s in mi-tochondria (Solish et al., 1988). Some evidence for amitochondrial cytochrome P450 in plants has beenpresented (Lindemann and Luickner, 1997), but noneof the large number of P450 genes in Arabidopsishave been characterized as mitochondrial, and a re-dox partner for this adrenodoxin in plant mitochon-dria remains elusive. The mitochondrial matrix iso-form of superoxide dismutase with Mn at its activesite was also identified (10), as was a methyltrans-

ferase involved in the final step of UQ synthesis (79).

Amino Acid Metabolism

A variety of enzymes associated with amino acidmetabolism were also identified. Ala and Asp amin-otransferases (111 and 154) allow the interconversionof TCA cycle intermediates with amino acid pools.Several broad range enzymes were also identifiedthat have the potential to be involved in a variety ofamino acid degradation pathways with an array ofdifferent substrates; these include the two aldehydedehydrogenases (158 and 16) and enoyl CoA hy-dratase (141). The aldehyde dehydrogenases function

broadly in amino acid and fatty acid metabolism tocatalyze the oxidation of aldehyde or oxo groups toform carboxylates through the reduction of NAD(P)to NAD(P) H. Interestingly, it was a putative alde-hyde dehydrogenase that was identified as the rf2nuclear restorer of the T-cytoplasm maize (Zea mays)cytoplasmic male sterility line (Cui et al., 1996). EnoylCoA hydratase catalyzes the removal of water from avariety of unsaturated acyl-CoAs in the degradationpathways of Val, Leu, iso-Leu, Lys, Trp, and -Ala.Methylmalonate semi-aldehyde dehydrogenase (27)acts downstream of enoyl coA hydratase in the ca-

tabolism of branched chain amino acids.

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Pyruvate is converted to Cys via the intermediate,3-mercaptopyruvate, and Ser is converted to Cys viaan acetyltransferase and Cys synthase. The presenceof 3-mercaptopyruvate sulfur transferase (76), Cyssynthase (54), and acetyltransferase-like proteins (11

and 130) suggests that such metabolic pathways may be in action in plant mitochondria. One of the sub-strates for 3-mercaptopyruvate sulfur transferase ishydrogen cyanide, which is also a potent inhibitor ofrespiration by the cytochrome pathway. It would beinteresting to consider if the operation of this meta-

bolic pathway may be correlated with the expressionof the alternative oxidase, which is insensitive tocyanide and thus able to maintain electron transport.Recently, a -cyano-Ala synthase, which also useshydrogen cyanide as a substrate and is structurallyrelated to the Cys synthases, was identified as amitochondrial protein in plants (Hatzfeld et al.,2000).

-Aminobutyric Acid (GABA) Shunt Metabolism

Several enzymes potentially involved in a GABAshunt of carbon intermediates between 2-oxoglutarateand succinate were also identified. Glu dehydroge-nase (40 and 43) forms Glu from 2-oxoglutarate to

begin the shunt and succinic semialdehyde dehydro-genase (26) concludes the shunt by the formation ofsuccinate. Recently, work by Busch and Fromm(1999) has identified that an Arabidopsis succinicsemialdehyde dehydrogenase was localized in mito-chondria. Previously, they had identified a Glu de-carboxylase in the cytosol (Snedden et al., 1996), buthave not found evidence of GABA aminotransferasegene(s), which are likely to code for mitochondrialprotein(s), for the completion of this putative shunt(Busch and Fromm, 1999). Several aminotransferaseswere identified in this study (154 and 111) and have

been annotated based on average homology to trans-ferases with known substrates. A range of other ami-notransferases with some homology to GABA and-Ala aminotransferases are also present in Arabi-dopsis protein DBs, and several have predicted mi-tochondrial presequences. Any of these proteins mayact as a GABA aminotransferase to complete this

stunt.

RNA Metabolism and Translation Apparatus

Proteins involved in mitochondrial RNA metabo-lism were also found in the Arabidopsis mitochon-drial proteome. Among these, a DEAD box RNAhelicase (163) was identified. A different protein ofthis family (AtSUV3) has recently been characterizedin Arabidopsis mitochondria (Gagliardi et al., 1999).The function of such proteins in plant mitochondriaremain unclear, but they are predicted to be involvedin transcript unfolding during, for example, tran-

script maturation or degradation processes. In yeast,

a homologous helicase, SUV3, is a component of themitochondrial 3- to 5-RNA degradosome (Margos-sian et al., 1996). The presence of at least two proteinsof this family in plant mitochondria might suggestthat different helicases could be transcript specific, or

alternatively, that they could be mechanism specific.For example, one could be involved in degradationand another in transcript maturation. Another pro-tein showing a typical Gly-rich domain, which ischaracteristic of RNA-binding proteins, was alsoidentified (2). This protein might be a component ofthe mitochondrial ribosome; it shows high similarity(data not shown) to the Arabidopsis mitochondrialribosomal protein rps19 (Sanchez et al., 1996). Severalmembers of the plant mitochondrial translational ap-paratus, the elongation factors Tu, G, and TS, werealso found in this analysis (47, 94, 168, 102, 103, and145). The mitochondrial EF-Tu protein has been char-acterized for Arabidopsis (Kuhlman and Palmer,1995) and recently for maize (Choi et al., 2000). How-ever, the putative functions of Q9SHD6 and O82505as elongation factors G and TS are only based onhomology to yeast mitochondrial proteins (Vambu-tas et al., 1991).

Comparisons of Targeting Prediction Programs UsingProteome Data

All the identified protein sequences were queriedthrough three intracellular targeting prediction pro-gram: TargetP, Psort, and MitoProt. TargetP andPsort assess a number of potential targeting destina-tions. They identified 54 and 49 protein sequences,respectively, as mitochondrial targeted based ontheir N-terminal region (Table II). In this analysis,TargetP was used in its winner-takes-all mode with-out setting a specificity cut-off for targeting. MitoProtonly predicts the likelihood of targeting to the mito-chondria based on the probability that a presentedsequence belongs to the group of known prese-quences based on 47 weighted characteristics. Mito-Prot identified 53 proteins as putatively mitochon-drial based on a probability cut-off of 0.85. Notably,all three prediction programs only agreed on a subset

of 32 of these protein sequences as being likely tar-geted to mitochondria. TargetP and Psort identified16 and 13 sequences, respectively, as chloroplast tar-geted, agreeing on only nine of this set. The disputedsequences in most cases were assessed as mitochon-drial by the other prediction program. This set in-cluded a range of well-known mitochondrial proteinssuch as subunits of succinly CoA ligase (3), pyruvatedehydrogenase complex (132), cytochrome c oxidase(151), and the mitochondrial elongation factor Tu (47,94, and 168). A remaining 20 to 30 proteins sequence,depending on the program, were not considered tocontain mitochondrial targeting sequences by the

three prediction programs. This set of protein se-




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quences included two aconitase protein sequences,the four prohibitin protein sequences, the two porinsequences, the TOM40 import pore protein sequence,and the adenylate kinase protein sequence. All theseproteins are known to be mitochondrial, but their

location (predominantly but not exclusively in theouter membrane and intermembrane space) does notrequire classical import by cleavable N-terminal ex-tensions. Of the 10 proteins of unknown functionidentified in this study, a total of six contained pu-tative N-terminal targeting sequences. The remainingfour (125, 143, 78, and 124) do not contain clearlyidentifiable targeting sequences; however, as wehave seen above, this may not preclude their mito-chondrial localization.

GRAVY Scores, Protein Solubility, and

Membrane Localization

A GRAVY score is a single value indication of theoverall hydropathicity of a protein sequence basedon the Kyte and Doolittle algorithmsthe more pos-itive the score the more hydrophobic the overall se-quence (Kyte and Doolittle, 1982). The addition ofthiourea as a chaotropic agent, the advent of newnonionic sulfobetaine detergents, and the use of thenoncharged reductant tributylphosphine have all becredited with allowing more hydrophobic proteins toenter two-dimensional gels for separated and analy-sis (Chevallet et al., 1998; Herbert et al., 1998; Rabil-

loud, 1998). To date, proteins with GRAVY scores ofapproximately 0.25 to 0.30 are considered to rep-resent the limit of these two-dimensional gel solubil-ity improvements (Herbert, 1999). Our analysis hasrevealed several proteins with positive GRAVYscores of 0.000 to 0.165; however, these are unlikelyproteins to be considered highly hydrophobic.They include malate dehydrogenase (56 and 202), the-subunit of succinyl CoA ligase (3), and the ATP-dependent protease (65), which were designated inthe soluble protein sample in Figure 2, as well as aperipheral membrane protein (26) and two unas-signed proteins (130 and 144). The integral mem-

brane proteins identified such as porin (57, 96, 14,and 128), prohibitins (13, 156, 166, and 169), andprocessing peptidases (25, 24, and 17) did not havepositive GRAVY scores. Our DMA localization study(Fig. 2; Table II) showed that many of the majorproteins shown to be soluble were subunits of theTCA cycle and the major HSPs of the 60 and 70classes. The subunits of the F

1Fo

ATP synthase wereall in the peripheral membrane class (Table II). Sooverall, although the localization of the identifiedproteins as soluble, peripheral membrane, or integralmembrane are largely in agreement with expectation,the GRAVY scores do not correlate well with these

DMA localizations.

DISCUSSION

A number of reports have presented two-dimensional gel arrays of mitochondria protein pro-files from pea (Humphery-Smith et al., 1992), potato(Solanum tuberosum; Colas des Francs-Small et al.,

1992), Arabidopsis (Davy de Virville et al., 1998), andmaize (Dunbar et al., 1997). Most of these reportshave highlighted changes that occur in this profile indifferent tissues from the same plant or during de-velopmental changes within the same cell type. How-ever, identifications of the different protein spots inthese profiles have been limited by the need forcross-reacting antibodies or sufficient material andfinance for N-terminal sequencing. The result has

been a rather limited understanding of many of themajor proteins that are found in the mitochondrialprofile. Recently, Prime et al. (2000) identified sixmitochondrial membrane-associated proteins in their

Arabidopsis organelle membrane preparations usinga combination of Edman degradation and MS/MS-based sequencing. Here, we show that the availabil-ity of full Arabidopsis genome sequence now enablesthe use of less expensive, high-throughput identifi-cations of even very low abundance plant proteinsfrom two-dimensional gel profiles by peptide massfingerprinting using MALDI-ToF MS data. Thisopens the way for full subproteome analyses inplants. We have used a cell suspension culture in ourexperiments, which has the advantages of lower cellheterogeneity, higher cytoplasmic contents, andgreater mass yields than a whole plant Arabidopsissystem. However, extension of this research intowhole plants and into specific differentiated plantorgans has the potential to identify other proteinsassociated with metabolic pathways not evident incell culture.

Identifying Functions in Plant Mitochondria

Based on the identifications we have obtained, ourattempts to minimize contamination of our mito-chondrial samples from plastid and peroxisomal ma-terial has been largely successful (Fig. 1). We havenot identified any photosystem proteins, componentsof the Calvin cycle, or major peroxisomal proteins(Table II). By analysis of the major proteins of Ara-

bidopsis mitochondria, we have begun to gain amore global appreciation of which proteins arepresent and thus which metabolic operations are pos-sible in plant mitochondria. This analysis also pro-vides insight into the relative abundance of differentproteins that may provide the framework for deter-mining turnover rates of different enzymes and theputative maximal fluxes of metabolic pathways. It isclear that from the results presented, the TCA cycle,the electron transport chain, and HSP60/HSP70sdominate the list of identifications (Table II) and thehighly abundant proteins visualized on the two-

dimensional gel profile (Fig. 3). The studies of these

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components in the plant mitochondrial research lit-erature are significant, no doubt aided by this abun-dance and the extensive knowledge of these meta-

bolic processes from mammals. Relatively little workhas focused on the amino acid catabolism by plant

mitochondria that is highlighted by the identifica-tions in Table II. In addition, we know little about thepotential presence of a GABA shunt, the possibilityof P450 reactions, or the role of the different classes ofchaperonins in plant mitochondria (Table II). Fur-thermore, more extensive investigations will be re-quired to identify the status and importance of theunknown function proteins identified in this study.

A variety of mitochondrial proteins that are knownto be present in significant abundance were not ob-served in this study. These include nearly all of theproteins encoded in the mitochondrial genome and arange of inner membrane carriers. In both of thesecases, the hydrophobicity and basic nature of the

protein sequences place them outside the resolvingability of current two-dimensional electrophoresis.The complex I, ATP synthase, cytochrome b/c

1com-

plex, and cytochrome oxidase subunits encoded inthe mitochondrial genome have GRAVY scoresgreater than 0.3 in almost every case, and the ribo-somal proteins in the mitochondrial genome aremore soluble in nature but have pI values of 10.4 to11.3. The inner membrane carriers have pI values inexcess of 9.5 and are also hydrophobic containing sixtransmembrane domains.

Peptide Mass Fingerprinting in Arabidopsis

Starting with a total of 170 spectra, we had 155high-quality spectra for DB searching. A total of 91spectra were matched to 77 different predicted pro-tein sequences, providing a percentage hit rate of59%. In the set of 77 protein sequences, only 11%were unknown proteins based on sequence compar-ison searches. In yeast and bacterial systems, peptidemass fingerprinting has yielded an approximately90% hit rate (Shevchenko et al., 1996), and 30% to40% of open reading frames (ORFs) identified bygenomic sequencing are assigned as unknown func-tion by sequence comparisons (The Arabidopsis Ge-nome Initiative, 2000). Although our data are onlypreliminary, an explanation of what lies behind thesestatistics seen in this Arabidopsis study is required tofurther our analysis of the mitochondrial proteome ofplants.

The inability to match 40% of spectra to predictedORFs is likely to be due to at least two factors. First,post-translational modifications to proteins will al-ter the apparent masses of peptides and precludematching to DB entries. The presence of such mod-ifications is evident from the differences in apparentmolecular mass and pI that were recorded for pro-tein spots that were matched to the same gene prod-

uct (Table II). Such modifications can be recognized

by MS/MS-based analysis of peptide sequences,and sequence tag information afforded by these MStechnologies will help to identify heavily modifiedproteins in the future. Second, matching to trans-lated ORFs requires that intron-exon boundaries

have been accurately determined, are in frame, andthat all ORFs in the genomic sequence have beenrecognized. Searches of redundant DBs of translatedORFs such as GENpept and TE readily illustratethat intron-exon boundaries are not always identi-fied accurately in Arabidopsis sequences. Thisphenomenon will be exacerbated in the case of un-known function proteins where sequence align-ments to characterized orthologs do not provide aready check of these splicing sites and frame shifts.This might explain the low overall hit rate and theeven lower hit rate on unknown function translatedORFs in our data (Table II). A solution to this prob-lem would be searches of peptide mass data not

only against predicted translated ORFs, but alsoagainst six frame translations of the full genomicsequence of Arabidopsis. This would not only im-prove peptide mass fingerprinting searches, but willalso act as a tool for the improvement of Arabidop-sis ORF identification and correct construction fromraw genomic sequence to supplement the use ofexpressed sequence tag sequence.

Future Mitochondrial Proteome Prediction

If the plant mitochondrial proteome is defined as

the native proteins encoded on the mitochondrialgenome and all the proteins encoded in the nucleusthat could possibly be targeted to this organelle, thenits elucidation is not a trivial matter. Comparison ofMitoProt, Psort, and TargetP suggests that no oneprogram currently provides a complete, high confi-dence analysis of the nuclear encoded components ofthe plant mitochondrial proteome. The small use ofcharacterized plant mitochondrial proteins in the es-tablishment of these tools and the abundant evidenceof mitochondrial targeting by means other thanN-terminal extensions do exacerbate these problems.The experimental approach we have outlined hereprovides direct evidence of proteins that are local-ized in mitochondria. However, this experimentalproteome is limited to those proteins that accumulateto significant levels, that are expressed in the tissuesample used, and that are able to remain solubleduring sample handling and separation. Furtheranalysis of low-abundance proteins will also nodoubt reveal low levels of contamination of mito-chondrial samples with other cellular compartments.We are currently working toward reducing theseexperimental limitations in a number of ways. Theseparation of mitochondria into outer membrane, in-ner membrane, inter-membrane space, and matrixcompartments reduces the complexity and increases

the amount of low-abundance proteins. Comparison




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of the proteome across developmental stages andfollowing environmental stimuli will identify pro-teins that are not found under the current cell growthregime. Improvements in isoelectric focusing (IEF)/SDS-PAGE, the use of blue-native-PAGE/SDS-

PAGE, and the advent of non-gel based chromatog-raphy for protein separation will also help toimprove the separation of hydrophobic proteins andfurther the experimentally identifiable mitochondrialproteome, especially those components encoded inthe mitochondrial genome. As these experimentalapproaches are explored, they will then provide the

badly needed basic data for the establishment of highconfidence bioinformatic based detection of the fullmitochondrial proteome.

MATERIALS AND METHODS

Maintenance of Cell Culture

A heterotrophic Arabidopsis cell culture, establishedfrom callus of cv Erecta stem explants, has been maintainedfor over 9 years by weekly subculture. Media used for thiscell culture was Murashige and Skoog basal media supple-mented with 3% (w/v) Suc, 0.5 mg/L naphthaleneaceticacid, and 0.05 mg/L kinetin (May and Leaver, 1993). Thecell cultures were maintained in the dark at 22C in anorbital shaker (150 rpm). At 6 to 7 d, each flask (120 mL)contained 8 to 10 g fresh weight of cells, and growth wasapproximately in the middle of the log phase. Subculture of20 mL of culture to 100 mL of fresh media began the cycleagain.

Mitochondrial Isolation

A total of 1.0 to 1.2 L of 7-d cell culture was filteredthrough gauze to remove media and was then ground bymortar and pestle, 30 g at a time. Grinding of each 30-galiquot was performed in 100 mL of grinding medium (0.3m mannitol, 50 mm sodium pyrophosphate, 0.5% [w/v]

bovine serum albumin [BSA], 0.5% [w/v] polyvinyl-pyrrolidone-40, 2 mm EGTA, and 20 mm Cys, pH 8.0).Filtered cell extract was separated by centrifugation at1,000g for 5 min at 4C and the supernatant was centri-fuged again at 18,000g for 15 min. The resulted organellepellet was washed by repeating the 1,000 and 18,000g cen-trifugation steps. The final organelle pellet was resus-pended in mannitol wash buffer (0.3 m mannitol, 0.1%[w/v]) BSA, and 10 mm TES (N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid]-NaOH, pH 7.5) andloaded onto a Percoll step gradient consisting of 1:4:2 ratio,

bottom to top, of 40% Percoll:23% Percoll:18% Percoll inmannitol wash buffer. The gradients were centrifuged for45 min at 40,000g, and mitochondria were present as anopaque band at the 23%:40% interface. This band wasaspirated, concentrated, and washed by centrifugation at15,000g for 15 min and was then loaded onto a self-formingPercoll gradient containing 28% Percoll in Suc wash buffer(0.3 m Suc, 0.1% [w/v] BSA, and 10 mm TES-NaOH, pH

7.5). After centrifugation at 40,000g for 30 min, mitochon-

dria banded near the top of the gradient and peroxisomalmaterial banded near the bottom of the gradient. The mi-tochondrial band was aspirated and again washed andconcentrated by two centrifugation steps at 15,000g for 15min in mannitol wash buffer.

Purity Measurements

Fractions of 1 mL each were collected from top to bottomof the Percoll step gradient and the Percoll self-forminggradient. The activities of the following marker enzymeswere measured in each fraction: cytochrome c oxidase (mi-tochondrion), catalase (peroxisome), alkaline pyrophos-phatase (plastid), and alcohol dehydrogenase (cytosol). En-zyme assays were as detailed in the following references:cytochrome c oxidase and catalase (Neuberger, 1985), alka-line pyrophosphatase (Gross and ap Rees, 1986), and alco-hol dehydrogenase (Smith and ap Rees, 1979).

Respiratory Measurements

Oxygen consumption was measured in a Clark-type ox-ygen electrode in 1 mL of reaction medium containing 0.3m mannitol, 10 mm TES-KOH, pH 7.5, 5 mm KH2PO4, 10mm NaCl, 2 mm MgSO4, and 0.1% (w/v) BSA. Pyruvate (5mm), malate (0.5 mm), succinate (10 mm), NADH (1 mm),ADP (0.5 mm), KCN (0.5 mm), and nPG (0.05 mm) wereadded as indicated to modulate oxygen consumption rates.Cytochrome c oxidase activity was measured as ascorbate(5 mm), cytochrome c (25 m)-dependent oxygen consump-tion in the presence of 0.05% (w/v) Triton X-100. Outermembrane integrity was assayed as the latency of cyto-chrome c oxidase activity (Neuberger, 1985),

Fractionation of Mitochondria

Mitochondria proteins were fractionated into soluble,membrane, integral membrane, and peripheral membranesamples. Approximately 10 mg of mitochondrial proteinwas incubated in 1 mL of 20 mm TES (pH 7.5), freeze-thawed in liquid nitrogen three times, and centrifuged for25 min at 20,000g. The supernatant represented solubleproteins. One-half of the pellet was retained as the totalmembrane fraction. The other one-half of the pellet wasresuspended in 1 mL of 100 mm Na2CO3 (pH of approxi-

mately 12), incubated for 20 min on ice, and again centri-fuged for 25 min at 20,000g (Fujiki et al., 1982). The pelletwas retained as integral membrane proteins, and the su-pernatant contained the peripheral membrane proteins.Protein samples were prepared for two-dimensional elec-trophoresis and loaded relative to the amount of totalmitochondrial protein from which each fraction was de-rived to allow direct comparisons between whole mito-chondrial samples and the different fractions.

Two-Dimensional Gel Electrophoresis

Mitochondria protein samples (500 g) were acetone-

extracted by the addition of acetone to a final concentration

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of 80% (v/v) at 80C, samples were stored at 20C for4 h, and then were then centrifuged at 20,000g for 15 min.The pellets were resuspended in an IEF sample buffer con-sisting of 6 m urea, 2 m thiourea, 2% (w/v) CHAPS {3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate}, 2%(v/v) ampholytes (310), 2 mm tributylphosphine, and0.001% (w/v) bromphenol blue. Aliquots of 330 L wereused to re-swell dried 180-mm, pH 3 to 10 nonlinear im-mobilized pH gradient strips (Immobiline DryStrips, Am-ersham Pharmacia Biotech) overnight and then IEF wasperformed for 19.5 h, reaching a total of 49 KVh at 20 C ona flat-bed electrophoresis unit (Multiphor II, AmershamPharmacia Biotech). Immobilized pH gradient strips werethen transferred to an equilibration buffer consisting of 50mm Tris-HCl (pH 6.8), 4 m urea, 2% (w/v) SDS, 0.001%(w/v) bromphenol blue, and 100 mmmercaptoethanol andwere incubated for 20 min with rocking. The equilibratedstrips were then slotted into central single wells of 4%(w/v) acrylamide stacking gels above 0.1 18.5 20 cm,

12% (w/v) acrylamide, 0.1% (w/v) SDS-polyacrylamidegels. Strips were overlaid with 0.5% (w/v) agarose in SDS-PAGE running buffer. Gel electrophoresis was performedat 25 mA per gel with circulating cooling and was com-pleted in 5 h. Proteins were visualized by colloidal Coo-massie (G250) staining. Molecular weight and pI standardsfrom Amersham Pharmacia Biotech were used to confirmfixed pH gradient positioning on first dimension separa-tion and to identify apparent molecular masses on seconddimension separation.

MALDI-ToF MS for Peptide Fingerprint Analysis

The majority of the in-gel digestion and MS was per-formed as a service by The Australian Proteome AnalysisFacility (Sydney, Australia) using a Micromass ToFSpec 2Emachine. Additional analyses were performed by the au-thors with the aid of Dr. Richard Lipscombe (The Univer-sity of Western Australia, Perth, Australia). Protein spots ofinterest were excised from gels and placed in wells of a96-titer plate. Destain solution (50% [v/v] acetonitrile, 25mm NH4HCO3) was added (50 L) to samples for 45 min,removed, and replenished once. Destained gel slices weredried at 50C for 20 min and digested at 37C in 10 L of25 mm NH4HCO3 containing 12.5 g/mL trypsin over-night. Acetonitrile (10 L) containing 1% (v/v) trifluoro-

acetic acid was added to each gel slice and incubated for 15min. Supernatant aliquots of 1 L were added directly tomatrix (-cyano-4-hydroxycinnamic acid [8 mg/mL], 50%[v/v] acetonitrile, and 1% [v/v] trifluoroacetic acid) and airdried onto a MALDI plate for MS analysis using anVoyager-DE Pro (Applied Biosystems, Foster City, CA).

Identification of DB Entry Matches andSequence Analysis

MALDI-ToF MS analyses provided a set of 15 to 30 pep-tide masses from each trypsinated protein sample. Thesemasses were used, via MS-Fit-based software, to identify

hits in translated GB, TE, and SWISS-PROT libraries, as

well as in the The Arabidopsis Information Resource-translated ORFs of Arabidopsis. Matching was performedat 50 ppm from the input masses and hits were assessed

by peptide number matching (516), coverage (typicallygreater than 25%), and the Molecular Weight Search score.All data were then cross-matched by comparison of thepredicted molecular weight and pI of the predicted pro-tein with the observed molecular mass and pI of the excisionsite on two-dimensional gels. TargetP (http://www.cbs.dtu.dk/services/TargetP/), MitoProt (http://www.mips.

biochem.mpg.de/cgi-bin/ proj/ medgen/mitofilter), andPsort (http://psort.nibb.ac.jp/) predictions, and GRAVYscores were determined using full-length predicted proteinsequences. Molecular mass, pI, and GRAVY scores weredetermined using the ProtParam program on an ExPASywebsite (http://au.expasy.org).

ACKNOWLEDGMENTS

This research has been facilitated by access to the Aus-tralian Proteome Analysis Facility established under theAustralian Governments Major National Research Facili-ties Program. We acknowledge the kind help of Dr. Rich-ard Lipscombe with MALDI-ToF analysis.

Received April 23, 2001; returned for revision May 31, 2001;accepted August 20, 2001.

LITERATURE CITED

Bartoli C, Pastori GM, Foyer C (2000) Ascorbate biosyn-thesis in mitochondria is linked to the electron transportchain between complexes III and IV. Plant Physiol 123:335343

Busch KB, Fromm H (1999) Plant succinic semialdehydedehydrogenase: cloning, purification, localization in mi-tochondria, and regulation by adenine nucleotides. PlantPhysiol 121: 589597

Chevallet M, Santoni V, Poinas A, Rouquie D, Fuchs A,Kieffe S, Rossignol M, Lunardi J, Garin J, Rabilloud T(1998) New zwitterionic detergents improve the analysisof membrane proteins by two-dimensional electrophore-sis. Electrophoresis 19: 19011909

Choi K, Roh K, Kim J, Sim W (2000) Genomic cloning andcharacterization of mitochondrial elongation factor Tu(EF-Tu) gene (tufM) from maize (Zea mays L.). Gene 257:233242

Claros MG, Vincens P (1996) Computational method topredict mitochondrially imported proteins and their tar-geting sequences. Eur J Biochem 241: 779786

Colas des Francs-Small C, Ambard-Bretteville F, DarpasA, Sallantin M, Huet J-C, Pernollet J-C, Remy R (1992)Variation of the polypeptide composition of mitochon-dria isolated from different potato tissues. Plant Physiol98: 273278

Cui X, Wise R, Schnable P (1996) The rf2 nuclear restorerof male-sterile T-cytoplasm maize encodes a putative

aldehyde dehydrogenase. Science 272: 13341336




16/17

Davy de Virville J, Alin M-F, Aaron Y, Remy R, Guillot-Salomon T, Cantrel C (1998) Changes in functionalproperties of mitochondria during growth cycle of Ara-bidopsis thaliana cell suspension cultures. Plant PhysiolBiochem 36: 347356

Day D, Neuberger M, Douce R (1985) Biochemical char-acterization of chlorophyll-free mitochondria from pealeaves. Aust J Plant Physiol 12: 219228

Dunbar B, Elthon T, Osterman J, Whitaker B, Wilson S(1997) Identification of plant mitochondrial proteins: aprocedure linking two-dimensional gel electrophoresisto protein sequencing from PVDF membranes using afastblot cycle. Plant Mol Biol Rep 15: 4661

Emanuelsson O, Nielsen H, Brunak S, von Heijne G(2000) Predicting subcellular localization of proteins

based on their N-terminal amino acid sequence. J MolBiol 300: 10051016

Fujiki Y, Fowler S, Shio H, Hubbard AL, Lazarow PB(1982) Polypeptide and phospholipid composition of the

membrane of rat liver peroxisomes: comparison withendoplasmic reticulum and mitochondrial membranes.

J Cell Biol 93: 103110Gagliardi D, Kuhn J, Spadinger U, Brennicke A, Leaver

C, Binder S (1999) An RNA helicase (AtSUV3) is presentin Arabidopsis thaliana mitochondria. FEBS Lett 458:337342

Gangloff S, Marguet D, Lauquin GM (1990) Molecularcloning of the yeast mitochondrial aconitase gene(ACO1) and evidence of the synergistic regulation ofexpression by glucose plus glutamate. Mol Cell Biol 10:35513561

Gray M, Burger G, Lang (1999) Mitochondrial evolution.

Science 283: 14761481Gross T, ap Rees T (1986) Alkaline inorganic pyrophos-

phatase and starch synthesis in amyloplasts. Planta 167:140145

Gueguen V, Macherel D, Jaquinod M, Douce R, Bour-guignon J (2000) Fatty acid and lipoic acid biosynthesisin higher plant mitochondria. J Biol Chem 275: 50165025

Halperin T, Zheng B, Itzhaki H, Clarke AK, Adam Z(2001) Plant mitochondria contain proteolytic and regu-latory subunits of the ATP-dependent Clp protease.Plant Mol Biol 45: 461468

Hatzfeld Y, Maruyama A, Schmidt A, Noji M, Ishizawa K,Saito K (2000) -Cyanoalanine synthase is a mitochon-drial cysteine synthase-like protein in spinach and Ara-

bidopsis. Plant Physiol 123: 11631171Herbert B (1999) Advances in protein solubilisation for two-

dimensional electrophoresis. Electrophoresis 20: 660663Herbert BR, Molloy MP, Gooley AA, Walsh BJ, Bryson

WG, Williams KL (1998) Improved protein solubility intwo-dimensional electrophoresis using tributyl phos-phine as reducing agent. Electrophoresis 19: 845851

Humphery-Smith I, Colas des Francs-Small C, Ambart-Bretteville F, Remy R (1992) Tissue-specific variation ofpea mitochondrial polypeptides detected by computer-ized image analysis of two-dimensional electrophoresisgels. Electrophoresis 13: 168172

Jenner H, Winning B, Millar AH, Tomlinson K, Leaver

CJ, Hill SA (2001) NAD malic enzyme and the control of

carbohydrate metabolism in potato tubers. Plant Physiol126: 11391149.

Kuhlman P, Palmer J (1995) Isolation, expression, andevolution of the gene encoding mitochondrial elongationfactor Tu in Arabidopsis mitochondria. Plant Mol Biol 29:10571070

Kyte J, Doolittle R (1982) A simple method for displayingthe hydropathic character of a protein. J Mol Biol 157:105132

Laloi M (1999) Plant mitochondrial carriers: an overview.Cell Mol Life Sci 56: 918944

Lindemann P, Luickner M (1997) Biosynthesis of pregnanederivatives in somatic embryos of Digitalis lanata. Phyto-chemistry 46: 507513

Lutziger I, Oliver DJ (2000) Molecular evidence of aunique lipoamide dehydrogenase in plastids: analysis ofplastidic lipoamide dehydrogenase from Arabidopsisthaliana. FEBS Lett 484: 1216

Margossian S, Li H, Zassenhaus H, Butow R (1996) The

DExH box protein Suv3p is a component of a yeastmitochondrial 3-to-5 exoribonuclease that suppressesgroup I intron toxicity. Cell 84: 199209

May MJ, Leaver CJ (1993) Oxidative stimulation of gluta-thione synthesis in Arabidopsis thaliana suspension cul-tures. Plant Physiol 103: 621627

Nakai K, Kanehisa M (1992) A knowledge base for pre-dicting protein localization sites in eukaryotic cells.Genomics 14: 897911

Neuberger M (1985) Preparation of plant mitochondria,criteria for assessment of mitochondrial integrity andpurity, survival in vitro. In R Douce, D Day, eds, HigherPlant Cell Respiration. Springer-Verlag, Berlin, pp 724

Nijtmans LGJ, de Jong L, Sanz MA, Coates PJ, Berden JA,Back JW, Muijsers AO, van der Spek H, Grivell LA(2000) Prohibitins act as a membrane-bound chaperonefor the stabilization of mitochondrial proteins. EMBO J19: 24442451

Peltier J-B, Friso G, Kalume D, Roepstorff P, Nilsson F,Adamska I, van Wijk K (2000) Proteomics of the chlo-roplast: systematic identification and targeting analysisof lumenal and peripheral thylakoid proteins. Plant Cell12: 319341

Peyret P, Perez P, Alric M (1995) Structure, genomic orga-nization, and expression of the Arabidopsis thaliana acon-itase gene: plant aconitase show significant homologywith mammalian iron-responsive element-binding pro-tein. J Biol Chem 270: 81318137

Prime T, Sherrier D, Mahon P, Packman L, Dupree P(2000) A proteomic analysis of organelles from Arabidop-sis thaliana. Electrophoresis 21: 34883499

Rabilloud T (1998) Use of thiourea to increase the solubil-ity of membrane proteins in two-dimensional electro-phoresis. Electrophoresis 19: 758760

Rasmusson AG, Svensson AS, Knoop V, Grohmann L,Brennicke A (1999) Homologues of yeast and bacterialrotenone-insensitive NADH dehydrogenases in highereukaryotes: two enzymes are present in potato mito-chondria. Plant J 20: 7987

Rebeille F, Macherel D, Mouillon JM, Garin J, Douce R

(1997) Folate biosynthesis in higher plants: purification

Millar et al.



17/17

and molecular cloning of a bifunctional 6-hydroxy-methyl-7,8-dihydropterin pyrophosphokinase/7,8-dihy-dropteroate synthase localized in mitochondria. EMBO J16: 947957

Saisho D, Nambara E, Naito S, Tsutsumi N, Hirai A,Nakazono M (1997) Characterization of the gene familyfor alternative oxidase from Arabidopsis thaliana. PlantMol Biol 35: 585596

Sanchez H, Fester T, Kloska S, Schroder W, Schuster W(1996) Transfer of rps19 to the nucleus involves the gainof an RNP-binding motif which may functionally replaceRPS13 in Arabidopsis mitochondria. EMBO J 15:21382149

Santoni V, Rouquie D, Doumas P, Mansion M, Boutry M,Degand H, Dupree P, Packman L, Sherrier J, Prime T etal. (1998) Use of a proteome strategy for tagging proteinspresent at the plasma membrane. Plant J 16: 633641

Shevchenko A, Jensen ON, Podtelejnikov AV, SaglioccoF, Wilm M, Vorm O, Mortensen P, Boucherie H, Mann

M (1996) Linking genome and proteome by mass spec-trometry: large-scale identification of yeast proteins fromtwo-dimensional gels. Proc Natl Acad Sci USA 93:1444014445

Sjoling S, Glaser E (1998) Mitochondrial targeting pep-tides in plants. Trend Plant Sci 3: 136140

Smith A, ap Rees T (1979) Pathways of carbohydrate fermen-tation in the roots of marsh plants. Planta 146: 327334

Snedden WA, Koutsia N, Baum G, Fromm H (1996) Acti-vation of a recombinant petunia glutamate decarboxyl-ase by calcium/calmodulin or by a monoclonal antibodywhich recognizes the calmodulin binding domain. J BiolChem 271: 41484153

Solish S, Picado-Leonard J, Morel Y, Kuhn R, MohandasT, Hanukoglu I, Millar W (1988) Human adrenodoxinreductase: two mRNAs encoded by a single gene onchromosome 17cen to q25 are expressed on steroidogenictissues. Proc Natl Acad Sci USA 85: 71047108

Steglich G, Neupert W, Langer T (1999) Prohibitins regu-late membrane protein degradation by the m-AAA pro-tease in mitochondria. Mol Cell Biol 19: 34353442

The Arabidopsis Genome Initiative (2000) Analysis of thegenome sequence of the flowering plant Arabidopsis thali-

ana. Nature 408: 796815Vambutas A, Ackerman S, Tzagoloff A (1991) Mitochon-

drial translational-initiation and elongation factors inSaccharomyces cerevisiae. Eur J Biochem 201: 643652


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