NEWS AND VIEWS

www.nature.com/naturebiotechnology • MARCH 2003 • VOLUME 21 • nature biotechnology

What is the set of proteins that constitute anorganelle in its entirety? Having the answerto this question is the dream of many cellbiologists. The advent of the ‘omics’ era haschanged the dream to an experimentallytractable problem. Several systematicapproaches are being pursued to define thecomplements of proteins associated withparticular intracellular structures or func-tions. Two studies published in this issue1,2

hold the promise that the combination ofbiochemical and genetic approaches will beextremely powerful to obtain a complete pic-ture of the mitochondrial proteome in thenear future.

The discovery of mitochondria as ubiqui-tous and defined entities of nucleated cellsdates back to the end of the 19th century.Some 50 years ago, mitochondria were rec-ognized as the power plants of the cell. In thelate sixties, mitochondrial DNA was discov-ered, and in the eighties the human mito-chondrial genome was sequenced. It becameclear that this genome encodes only a hand-ful of mitochondrial proteins (i.e., 8 in yeastand 13 in human), whereas the vast majorityof proteins are encoded by the nuclear DNA,synthesized in the cytosol and imported intothe organelle. At the same time, mitochondr-ial diseases were found to be due to muta-tions both in the mitochondrial genomeand, more recently, in the nuclear genome.In recent years, research on the roles of mito-chondria in apoptosis, aging, and the patho-genesis of several diseases (includingParkinson’s, Alzheimer’s and cancer) hasgained much interest. One can foresee thatelucidation of the mitochondrial proteomewill be the next big step toward an in-depthunderstanding of this complex organellebecause it will be the basis for the moleculardissection of known and novel mitochondri-al functions in the coming decades.

How many different proteins make up amitochondrion? The most comprehensivestudy performed so far was aimed at localiz-

ing the proteome in yeast cells. In a large-scale study, 2744 epitope-tagged yeast pro-teins covering roughly 45% of the proteomewere immunolocalized. A mitochondriallocation was found for 332 proteins, repre-senting 13% of the set analyzed. From thisanalysis, it can be estimated that yeast mito-chondria contain about 800 distinct pro-teins3. Given the complexity of differentiatedmammalian cells, the number of distinctproteins in human mitochondria is likely tobe considerably higher; perhaps between1000 and 2000, or even higher.

The present paper by Taylor et al. 1 pur-sues a biochemical approach to obtain themost complete catalog of mitochondrialproteins reported so far. Highly purifiedmitochondria isolated from human heartwere solubilized with a mild detergent, pro-

tein complexes were partially separated bysucrose gradient centrifugation, and pro-teins were resolved by one-dimensional gelelectrophoresis. Mitochondrial proteinswere identified by mass spectrometry com-bined with rigorous bioinformatic analysis.Thus, a total of 615 mitochondrial or mito-chondria-associated proteins were identi-fied. These include a significant number ofpotentially new mitochondrial proteins, thebiochemical functions of which remain to bedefined.

Several similar studies aimed at a system-atic identification of mitochondrial proteinsby proteomic approaches have been report-ed. The most comprehensive study reportedso far was on rat liver mitochondria andyielded a list with 192 proteins4. Remarkably,Taylor et al. were able to triple the size of thatlist. There is no doubt that this is a majorachievement. However, at least half of themitochondrial proteome remains unknown.The elucidation of the complete set of mito-chondrial proteins will certainly be sped upby the combination of several approachescoming from different disciplines. Thus, theaccompanying paper by Umezawa and col-leagues2 is very timely. These latterresearchers developed an elegant geneticscreening method that holds the potential toidentify novel mitochondrial proteins on alarge scale from cDNA libraries.

Their approach is based on reconstitutionof a split green fluorescent protein (GFP)reporter in mitochondria. Sequences fromcDNA libraries are randomly fused to theamino-terminal half of GFP. If the expressedfusion protein contains targeting informationfor the mitochondrial matrix, the GFP moietywill meet its carboxy-terminal counterpartthere. Full-length GFP is reconstituted in thematrix by a protein splicing element presentin both reporter constructs. This methodolo-gy has the advantage that only mitochondria-positive clones yield a fluorescence signal.These clones can be isolated by automatedcell sorting, a technique that allows a wide netto be cast. Relevant genes are subsequentlyidentified by molecular genetic techniques.Although the system has not yet been appliedin a comprehensive manner, the authorsdemonstrate its suitability for rapid identifi-cation of novel proteins containing mito-chondrial targeting information.

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Figure 1. Two roads towards an elucidation ofthe mitochondrial proteome. A biochemical1 anda genetic2 approach promise to define thecomplement of proteins that make up amitochondrion.

‘Omics’ of the mitochondrionTwo complementary proteomics approaches promise to move us closer to definition of the completecomplement of proteins that make up a mitochondrion.

Benedikt Westermann and Walter Neupert

Benedikt Westermann is a group leader andWalter Neupert is a professor and director atthe Institut für Physiologische Chemie,Universität München, Butenandtstr. 5, 81377München, Germany (neupert@bio.med.uni-muenchen.de).

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nature biotechnology • VOLUME 21 • MARCH 2003 • www.nature.com/naturebiotechnology

NEWS AND VIEWS

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Both biochemistry and genetics haveproven to be instrumental in identifyingmitochondrial components. However, somelimitations are associated with the strategiesdiscussed above. Proteins of low abundancemight be missed because they are present inamounts too low for identification by gelelectrophoresis and mass spectrometry, andtheir genes might be underrepresented incDNA libraries. Furthermore, both methodsmight yield a significant number of false-positive candidates; mitochondrial prepara-tions may be contaminated by otherorganelles, or some sequences taken out oftheir normal context may artificially target apassenger protein to the mitochondrion.The genetic system, at least in its presentform, requires that the GFP fusion protein isexposed to the matrix and thus fails to iden-tify outer membrane and intermembranespace proteins as well as certain inner mem-brane proteins.

Other recent efforts to achieve a genome-wide identification of mitochondrial com-ponents have relied on the analysis of yeastmutant collections defective in mitochondr-ial functions5,6 or on the changes of the tran-scription profiles dependent on respiratoryactivity7,8. These studies, however, arerestricted to proteins involved in oxidativephosphorylation and cannot discriminatebetween mitochondrial proteins and factorsthat are indirectly involved, (e.g., nucleartranscription factors).

Although comprehensive biochemicaland genetic approaches combined withcomputational predictions are providing amore complete picture of the mitochondrialproteome, important challenges remain.First, more information must come fromcomparative proteomics: analysis of the pro-teomes of different organs will be importantin correlating different structures with dif-ferent sets of proteins. And second, mito-chondrial proteomes need to be studiedunder the various physiological or patholog-ical conditions to teach us more about theroles of mitochondria in health and disease.At the same time, it will be important toassign biochemical functions to the manynewly identified proteins. Clearly, knowingthe complete set of proteins that constitute amitochondrion will open a new era of mito-chondrial biology.

1. Taylor, S.W.et al.Nat.Biotechnol. 21, 281–286 (2003).2. Ozawa, T., Sako, Y., Sato, M., Kitamura, T., &

Umezawa, Y. Nat. Biotechnol. 21, 287–293 (2003).3. Kumar, A. et al. Genes Dev. 16, 707–719 (2002).4. Fountoulakis, M., Berndt, P., Langen, H., & Suter, L.

Electrophoresis 23, 311–328 (2002).5. Steinmetz, L.M. et al. Nat. Genet. 31, 400–404

(2002).6. Dimmer, K.S. et al. Mol. Biol. Cell 13, 847–853 (2002).7. Hughes, T.R. et al. Cell 102, 109–126 (2000).8. Epstein, C.B. et al. Mol. Biol. Cell 12, 297–308 (2001).

Type 2 diabetes is one of the world’s mostcommon diseases, affecting more than 150million people across the globe, and thisnumber is predicted to double within thenext 15 years. No cure is available for type 2diabetes, and management of the diseasetypically involves diet control, exercise,home blood glucose testing, and in somecases, medication with recombinant insulinor oral pharmaceuticals. The application ofgenomics-based target discovery to type 2diabetes has long promised to provide newtargets for medical intervention. In thisissue, Chen et al.1 apply such an approach tofunctional screening for secreted proteinswith therapeutic potential in type 2 dia-betes. Their screens reveal bone morpho-genetic protein-9 (BMP-9) as a promisingtarget for further investigation in diabetesdrug discovery.

The epidemic increase in the prevalenceof type 2 diabetes is attributed to a syner-gism between genetic predisposition andobesity common in affluent western soci-ety2. However, the root cause of the geneticpredisposition has thus far eluded most sci-entific enquiry. In type 2 diabetes, elevatedglucose concentration (chronic hyper-glycemia) results from impaired secretionof insulin and insulin resistance in targettissues like muscle and liver. As early as themid-19th century, Claude Bernard suggest-ed that the liver plays a central role in thisscenario3. A putative hormone, hepaticinsulin-sensitizing substance (HISS), whichis released from the liver and enhances glu-cose uptake in peripheral tissues, has beendiscussed and debated for half a decade4.The nature of this “hormone” has, however,remained obscure. The BMP-9 moleculeidentified by Chen et al. in this issue repre-sents a putative HISS.

To identify factors that could influencekey steps involved in diabetic pathogenesis,

Chen et al. first searched for secreted pro-teins among more than 3 million expressed-sequence tags (ESTs) from 1,000 differentcDNA libraries in the Human GenomeSciences (Rockville, MD) database. Theyscanned all the resulting open readingframes (ORFs) starting with an ATG for thepresence of an N-terminal secretory signalpeptide using two different bioinformaticalgorithms: a hidden Markov model and theSignalP classifier program. ComplementaryDNAs of the ESTs that scored positive byboth methods were sequenced to confirmthat they were full-length. Approximately8,000 sequences containing complete codingregions, starting with a putative signal pep-tide, were then assessed in a functionalscreening program (Fig. 1).

In the next step, the 8,000 secreted pro-teins were transiently transfected intohuman embryonic kidney (HEK) 293 cellsand the supernatants tested in high-throughput cell-based assays for their abilityto modulate glucose metabolism. Theresearchers assessed the proteins for a role insuppression of hepatic glucose productionby screening for their ability to inhibit theexpression of a key rate-limiting enzyme ingluconeogenesis, phosphoenolpyruvate car-boxykinase (PEPCK) in liver rat hepatomaH4IIe cells. They also tested their capacity toactivate the serine/threonine kinase Akt(which stimulates glycogen synthesis inmuscle) and to stimulate the transcription oftwo proteins essential for fat synthesis, malicenzyme (ME) and fatty acid synthase (FAS).

BMP-9 scored as a hit in the assays, withan observed effect that was comparable tothat seen with insulin. Moreover, purifiedrecombinant BMP-9 was shown to cause asustained lowering of plasma glucose con-centrations in normal (C57BL/6) and dia-betic (db/db) mice. In normal mice, therewas a delayed dose-dependent response,with a hypoglycemic effect 24 hours aftertreatment, and in the diabetic mice glucoseconcentrations declined within the first 30hours after treatment. In addition to mimic-king the action of insulin, BMP-9 also stimu-lated insulin release in Wistar and Zuckerdiabetic rats.

Bringing diabetes therapeutics to the bigscreenLarge-scale genomic screening of secreted proteins for therapeuticpotential in diabetes reveals a surprise—bone morphogeneticprotein-9.

Leif Groop

Leif Groop is a professor and department headat the Department of Endocrinology,University Hospital MAS, Lund University,20502 Malmö, Sweden(Leif.Groop@endo.mas.lu.se).

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