pam16 has an essential role in the mitochondrial protein import motor

A R T I C L E S

226 VOLUME 11 NUMBER 3 MARCH 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

More than 98% of mitochondrial proteins are encoded by nucleargenes and synthesized as precursor proteins on cytosolic polysomes1–7.Preproteins destined for the innermost mitochondrial compartment,the matrix, are directed by N-terminal targeting signals (presequences)across both mitochondrial membranes. The preproteins cross themembranes via two proteinaceous channels formed by the translocaseof the outer membrane (TOM complex) and the presequence translo-case of the inner membrane (TIM23 complex), respectively.

The presequence translocase consists of three integral inner-membrane proteins essential for cell viability: Tim50, Tim17 and thechannel-forming protein Tim23 (refs. 2,8–11). The membrane poten-tial (∆ψ) across the inner membrane drives the translocation of the N-terminal presequences across this membrane by activating the Tim23 channel and by exerting an electrophoretic effect on the positively charged presequences8,12–15.

The translocation of entire precursor polypeptides into the matrix,however, requires an additional energy-consuming machinery, thematrix-exposed import motor whose central component is the ATP-dependent heat shock protein 70 (mtHsp70)5,16–18. Three essentialsubunits of the PAM have been known for about a decade and theirfunction has been analyzed in detail: mtHsp70; the nucleotideexchange factor of the matrix, Mge1; and the peripheral inner-membrane protein Tim44, which serves as a binding site for mtHsp70,bringing it close to the protein import channel. Various models

describing the function of mtHsp70 have been based on the assump-tion that the import motor consists of only those three compo-nents5,15,18–20. However, a mechanistic understanding of PAMdepends on knowing the full complement of the factors involved.Typically, Hsp70 chaperones such as Kar2 (BiP) of the endoplasmicreticulum, cytosolic Hsp70s, and DnaK of bacteria cooperate withcochaperones of the J class17,21–24. Indeed, a fourth essential motorsubunit has recently been found, termed Pam18 (or Tim14). Pam18 isan inner-membrane protein with a classical J domain that stimulatesthe ATPase activity of mtHsp70 and is required for protein trans-location across the inner membrane25–27.

We investigated whether the PAM contains additional componentsand indeed found a fifth essential subunit. This subunit, Pam16, is anew type of motor component that is conserved among eukaryotes.Pam16 cooperates with Pam18 to promote the mtHsp70 reactioncycle. These findings suggest that protein translocation into the mito-chondrial matrix is driven by a multisubunit PAM machinery.

RESULTSPam16 associates with the presequence translocaseWe used a Saccharomyces cerevisiae strain containing a protein A–tagon Tim23 to isolate the presequence translocase and associated com-ponents9,25. Isolated mitochondria were lysed with the mild detergentdigitonin and subjected to affinity purification, leading to the isolation

1Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany. 2Fakultät für Biologie, UniversitätFreiburg, D-79104 Freiburg, Germany. 3Laboratoire propre du CNRS Université Pierre et Marie Curie, F-91190 Gif-sur-Yvette, France. 4Medizinisches Proteom-Center, Ruhr-Universität Bochum, D-44780 Bochum, Germany. 5Rudolf-Virchow-Center for Experimental Biomedicine, Universität Würzburg, D-97078 Würzburg,Germany. 6Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, California 92697-3900, USA. 7Present addresses: Departmentof Comparative Physiology, Evolutionary Biology Centre, Uppsala University, SE-75236 Uppsala, Sweden (M.L.) and Curacyte AG, D-80339 München, Germany(A.G.). 8These authors contributed equally to this work. Correspondence should be addressed to N.P. ([email protected]) or P.R.([email protected]).

Published online 15 February 2004; doi:10.1038/nsmb735

Pam16 has an essential role in the mitochondrial proteinimport motorAnn E Frazier1,2,8, Jan Dudek1,2,8, Bernard Guiard3, Wolfgang Voos1, Yanfeng Li1,2, Maria Lind1,7, Chris Meisinger1, Andreas Geissler1,7, Albert Sickmann4,5, Helmut E Meyer4, Virginia Bilanchone6, Michael G Cumsky6, Kaye N Truscott1, Nikolaus Pfanner1 & Peter Rehling1

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, thetranslocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not forprotein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with thepresequence translocase and for formation of a mtHsp70–Tim44 complex. Thus, Pam16 is a newly identified type of motorsubunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.

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A R T I C L E S

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 3 MARCH 2004 227

of Tim23, Tim17 and Tim50 (the TIM23 complex), and of the PAMsubunits mtHsp70, Tim44 and Pam18 (Fig. 1a, lane 1). Additionalprotein bands found in the affinity purification include fragments ofknown subunits of TIM23 and PAM, as well as the inner-membraneprotein ADP-ATP carrier (AAC). The highly abundant AAC probablyrepresents a contamination of the purified TIM23 complex9. Usingmass spectrometry, we identified a new protein of 16 kDa (Fig. 1a),termed Pam16 according to the Saccharomyces cerevisiae genome data-base (http://www.yeastgenome.org). Deletion of the open readingframe YJL104W encoding PAM16 is lethal to yeast cells (not shown),indicating that Pam16 has a crucial role in maintaining cell viability.The predicted primary structure of Pam16 contains 149 residues(Fig. 1b). S. cerevisiae Pam16 is well conserved among eukaryotes such as Neurospora crassa, Schizosaccharomyces pombe, Drosophilamelanogaster, Caenorhabditis elegans and Homo sapiens with aminoacid identities of 27–39% and similarities of 62–69% (Fig. 1b).Notably, Pam16 contains a region of ∼ 85 residues with substantialsimilarity to the classical J domain–containing proteins, which areknown as cochaperones of Hsp70s17,21–24. A comparison of this regionof Pam16 to the J domain of Pam18 revealed an amino acid identity of19% and a similarity of 48% (Fig. 1c). The three characteristic α-helical segments of J proteins were also found in Pam16 (Fig. 1c), asdetermined by structural predictions. However, Pam16 lacks the J-protein signature motif HPD (His-Pro-Asp; boxed in Fig. 1c).

The primary structure of Pam16 suggests the presence of an N-terminal mitochondrial targeting signal. The precursor of Pam16was synthesized and 35S-labeled in rabbit reticulocyte lysate. Uponincubation with isolated yeast mitochondria, Pam16 was transported

to a location where it was protected against externally added protease(Fig. 1d). This transport was blocked when the membrane potentialacross the inner membrane was dissipated (Fig. 1d, lane 12). On SDS-PAGE, imported Pam16 showed the same mobility as the precursorform in reticulocyte lysate (Fig. 1d), indicating that the mitochondrialtargeting signal is not proteolytically removed upon import. When theouter mitochondrial membrane was ruptured by hypotonic swellingafter the import reaction, imported Pam16 was still protected againstexternally added protease (Fig. 1e, lane 2). When the mitochondrialmembranes were lysed by detergent, Pam16 was digested by the protease (Fig. 1e, lane 3), indicating that Pam16 is protected againstprotease by the inner-membrane barrier.

To determine the localization of endogenous Pam16, we raised antibodies against the expressed protein. Pam16 remained protectedagainst protease in mitochondria and mitoplasts (swollen mito-chondria), like the matrix-exposed protein Tim44. Intermembranespace-exposed Tim23 served as an internal control for intact versusswollen mitochondria (Fig. 2a). When the matrix was opened by sonica-tion, Pam16 and Tim44 were fully accessible to the protease (Fig. 2a,lanes 8 and 9). In addition, we determined whether Pam16 is mem-brane-associated by separating the membrane fraction from the solublefraction after sonication. Pam16, like AAC and Tim44, remained in themembrane fraction, whereas the soluble matrix protein Mge1 wasreleased into the supernatant (Fig. 2b, lanes 1–3). Upon treatment ofmitochondria at alkaline pH, Pam16 was mostly extracted, whereas theintegral membrane protein AAC remained in the membrane sheets(Fig. 2b, lanes 4–6). This result is in agreement with the structural pre-diction that indicates a lack of hydrophobic transmembrane segments in

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cPam16 27 QAQAASQSVKVKQGATNASASRRGTGKGEYGGITLDESCKILNIEESKGDLNMDKINNINNRFNYLFLFEVNDKEKGGSFYLQSKVYRAAERLKWELAQR 115Pam18 84 KSKSISKGLNLNGGKSTTATAFLKGGFDPK--MNSKEALQILNLTEN--TLTKKKLKELKEVHRKIMIMLANHPDKGGSPFLATKINEAKDFLEKRGISK 168

I II

bSc Pam16 1 MAHRAFIQVIITGTQVFGKAFAEAYRQAASQSVKQGATNAS------RRGTGKGEYG---GITLDESCKILNIEESKG--DLNM 73Nc Pam16 1 MAYRLITQVVVVGSRVLGRAFAEAYKQAAASSQYQRAQQKN-----GNAATGRASLTS--GMTLDEACKILNVNKPADGTAANM 77Sp Pam16 1 MSLPRAVGRFIIVGSQVMSKAFVQAYKQMIANAAQQSTGQAA-----ASKSSTAVRRG---EMTIQEAGSILNIK-PES---LEE 73Hs Pam16 1 MAKYLAQIIVMGVQVVGRAFARALRQEFAASRAAADARG-------RAGHRSAAASNLSGLSLQEAQQILNVS-KLS-----P 70Dm Pam16 1 MAKYIAQIIVLGAQAVGRAFTKALRQEIAASQEAARRAGG-----GKQGDKSAESNLRTGMTLEEAKQILNIDDPKN-----V 73Ce Pam16 1 MPWRTALKVALAAGEAVAKALTRAVRDEIKQTQQAAARHAASTGQSASETRENANSNAKLGISLEESLQILNVKTPLN-----R 79

Sc Pam16 74 DKINNRFNYLFEVNDKEKGGSFYLQSKVYRAAERLKWELAQREKNAK-----AKAGDASTAKPPPNSTNSSGADNSASSNQ 149Nc Pam16 78 EEVMERFKRLFDANDPEKGGSFYLQSKVVRARERLEAEIKPKMEEKQ-----AEEEVKEGWNPKIYKDR 141Sp Pam16 74 GELEKRFQKMFEINDPKKGGSFYLQSKVFRAHEKLKSELDQKIQE--------QSPAKPTSSP 128Hs Pam16 71 EEVQKNYEHLFKVNDKSVGGSFYLQSKVVRAKERLDEELKIQ----------AQEDREKGQMPHT 125Dm Pam16 74 DAITKNYEHLFQVNERSKGGSFYIQSKVFRAKERLDHEIKAHEQPRSSNTEAAQDTAEESQSRSRQRR 141Ce Pam16 80 EEVEKHYEHLFNINDKSKGGTLYLQSKVFRAKERIDEEFGRIELK-------EEKKKEENAKTE 136

III

Figure 1 Pam16 is associated with the mitochondrial presequence translocase. (a) Colloidal Coomassie-stained SDS-PAGE of the isolated TIM23 complex.Bands were analyzed by LC-MS-MS. (b) Sequence alignments of predicted Pam16 proteins from S. cerevisiae (Sc), N. crassa (Nc), S. pombe (Sp),H. sapiens (Hs), D. melanogaster (Dm) and C. elegans (Ce). Black, identical residues in at least four of the proteins; bold print, similar residues. Thesequence corresponding to the HPD motif of J proteins is boxed. (c) Comparison of Pam16 with the Pam18 J domain. Helical segments conserved in J proteins are indicated by I–III. Box, HPD motif of Pam18 and corresponding sequence of Pam16. (d) 35S-labeled Pam16 was imported into yeastmitochondria for the indicated times in the presence or absence of a membrane potential (∆ψ). Where indicated, mitochondria were treated with proteinaseK after import. 35S-labeled Pam16 is shown for reference. Samples were analyzed by SDS-PAGE and digital autoradiography. (e) After import of 35S-labeledPam16 into mitochondria for 10 min, the organelles were left untreated, subjected to hypotonic swelling or lysed in 0.5% (w/v) Triton X-100. Samples weresubsequently treated with proteinase K where indicated and analyzed as in d. For comparison, 35S-labeled Pam16 was treated with proteinase K.

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the primary structure. In summary, Pam16 behaves as a peripheral mito-chondrial inner-membrane protein that is exposed to the matrix space.

To determine whether Pam16 is enriched in the Tim23-associatedfraction, the eluate of a Tim23 affinity purification from digitonin-lysed mitochondria was probed with specific antibodies. Pam16 wasspecifically present in the eluate from mitochondria carrying thetagged Tim23, like Pam18, mtHsp70 and Tim50, but was not asstrongly enriched as Tim17, which forms a very tight complex withTim23 (ref. 28) (Fig. 2c, lane 2). The second matrix Hsp70, Ssq1, andthe cochaperones Mdj1 and Mge1, were not found in the Tim23-associated fraction (Fig. 2c, lane 2). None of the proteins was presentin the mock eluate when wild-type mitochondria were subjected to theaffinity chromatography (Fig. 2c, lane 4), confirming the specificity ofPam16 copurification with the TIM23 complex.

We then investigated whether Pam16 is part of a functional trans-location machinery engaged in preprotein transport. The matrix-targeted model preprotein b2(167)∆-DHFR, consisting of an N-terminal segment of mitochondrial cytochrome b2 and the passen-ger protein dihydrofolate reductase, can accumulate in the TOM andTIM23 import channels simultaneously when the C-terminal DHFRmoiety is stabilized by the ligand methotrexate9,25,28,29. When theTOM complex is purified via a tagged Tom22, subunits of the TIM23complex and the import motor, such as Tim23, Tim50 and Tim44, arecopurified via the accumulated preprotein in a TOM–TIM–PAMsupercomplex25,29 (Fig. 2d, lane 5), whereas in the absence of the pre-protein, only the TOM complex is enriched30 (Fig. 2d, lanes 11 and23). Pam16 copurified with the supercomplex only in the presence ofaccumulated b2(167)∆-DHFR (Fig. 2d, lane 5), suggesting that it isassociated with a functional translocase machinery.

Pam16 is required for matrix protein translocationWe generated temperature-conditional yeast mutants of PAM16 byerror-prone PCR, then selected the mutant cells pam16-1 and pam16-3,which grew like wild-type cells at 24 °C on fermentable and nonfer-mentable media, but did not grow at 37 °C (Fig. 3a). When the mutantcells were grown first at 24 °C and then shifted to 37 °C, they accumu-lated nonprocessed mitochondrial preproteins in such amounts thatthey could be detected by western blotting, whereas the wild-type cellsdid not accumulate detectable amounts of nonprocessed preproteins(Fig. 3b). This finding raised the possibility that Pam16 was involved inprotein import into mitochondria.

For a detailed analysis of protein translocation into mitochondria,we conducted in vitro import experiments with radiolabeled prepro-teins and isolated mitochondria. To minimize indirect effects of themutations, the cells were grown at the permissive temperature of24 °C, and mitochondria were isolated and preincubated for 15 min at37 °C to induce the mutant phenotype. We used several matrix-targeted preproteins, including the β subunit of the mitochondrial F1-ATPase (F1β) (Fig. 3c), b2(167)∆-DHFR, and a longer b2-fusionprotein, b2(220)∆-DHFR (Fig. 3d). In addition, we used saturatingamounts of a purified and urea-denatured b2-fusion protein, b2(47)-DHFR (Fig. 3e). The import of all preproteins analyzed wassubstantially reduced in pam16-1 and pam16-3 mitochondria as compared with wild-type mitochondria.

To assess the specificity of the protein import defect in pam16mutant mitochondria, we analyzed the composition and function ofthe mutant mitochondria as compared with wild-type mitochondriaisolated from cells grown at the permissive temperature of 24 °C. Thesteady-state levels of various protein markers were similar betweenwild-type and mutant mitochondria (Fig. 4a), including subunits ofthe TIM23 complex and the PAM machinery, F1β, subunits of the

TIM22 translocase for carrier proteins of the inner membrane (Tim22and Tim54), AAC, subunits of the TOM complex (Tom70 andTom40), and the abundant outer membrane protein porin. An analy-sis by blue native PAGE revealed that the three mitochondrial translo-case complexes for protein import, TIM23 complex, TOM complexand TIM22 complex, were present in pam16 mutant and wild-typemitochondria (Fig. 4b). The membrane potential was assessed by fluorescence quenching with the dye 3,3′-dipropylthiadicarbocyanineiodide (DiSC3(5)) and was indistinguishable between wild-type andmutant mitochondria (Fig. 4c).

Carrier proteins that contain multiple internal targeting signals aretransported into the inner membrane by the TIM22 complex and notby the TIM23 complex1,3,6,31–33. The ∆ψ-dependent assembly pathwayof the radiolabeled dicarboxylate carrier was analyzed by blue nativePAGE9,34 (Fig. 4d). The efficiency of assembly in pam16 mutant mito-chondria was not reduced as compared with wild-type mitochondria,

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Figure 2 Pam16 is exposed to the matrix and present in a functionaltranslocase–preprotein supercomplex. (a) Before proteinase K treatment,mitochondria were left on ice, subjected to hypotonic swelling, or lysed bysonication. Samples were subjected to SDS-PAGE and western blotting.(b) Mitochondria were sonicated or extracted by alkaline treatment. Sampleswere left untreated (T) or centrifuged and analyzed as in a. S, supernatant;P, pellet. (c) Copurification of Pam16 with the presequence translocase.Mitochondria were lysed (control) and the TIM23 complex isolated.Irrelevant gel lanes were digitally excised. (d) b2(167)∆-DHFR was arrestedin mitochondria carrying Tom22His10 (Tom22 with a C-terminal His10-tag) in the presence of methotrexate (MTX). Mitochondria were lysed in digitoninand subjected to Ni-NTA agarose chromatography. Samples were analyzed by SDS-PAGE and western blotting. 10% load (control), 100% of wash and100% eluate are shown.

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indicating that the carrier transport pathway to the inner membranewas not disturbed.

We then studied the import of two preproteins that contain an N-terminal targeting signal and are inserted into the inner membraneby the TIM23 complex, but do not require the function of mtHsp70owing to a hydrophobic insertion signal after the N-terminal targetingsignal: the precursor of cytochrome c1 and a shortened construct ofcytochrome c oxidase subunit Va (CoxVa∆)26,35–38. Both preproteins areinserted into the inner membrane in a ∆ψ-dependent manner. The pre-cursor of cytochrome c1 is then proteolytically processed in two steps,via the matrix processing peptidase and the inner-membrane peptidase(Fig. 4e)39,40. The independence of CoxVa∆ import from mtHsp70 func-tion is shown by using the mtHsp70 mutant mitochondria, ssc1-3(ref. 38) (Fig. 4f, lanes 16 and 18). The import of both precursor pro-teins occurred with similar efficiency in wild-type mitochondria and inthe pam16 mutant mitochondria (Fig. 4e,f), demonstrating that theTIM23 complex of pam16 mutant mitochondria is functional.

We conclude that mitochondria from both pam16 mutants areselectively impaired in the translocation of preproteins into thematrix, whereas the insertion of proteins into the inner membrane viathe TIM23 complex or the TIM22 complex is still possible, suggesting

that Pam16 may be required for the functionof the matrix import motor PAM.

Pam16-Pam18 interaction promotes PAMreaction cycleTo obtain in vivo evidence for potential part-ners of Pam16, we tested whether the growthphenotype of pam16 mutant cells could besuppressed by overexpression of subunits of

the import motor or the TIM23 complex. Of all components tested,only overexpressed Pam18 allowed for the growth of pam16-1 mutantsat 37 °C, aside from overexpressed wild-type Pam16 itself (Fig. 5a).However, the suppression of the pam16-1 growth defect by Pam18 wasnot complete. This finding, together with the observations that neitherthe growth defect of pam16-3 mutant cells nor the lethal phenotype ofdeletion of PAM16 were suppressed by overexpression of Pam18 (datanot shown), indicate that the inner-membrane J protein Pam18 canonly partially suppress defects in Pam16. The matrix J protein Mdj1did not suppress pam16 mutant cells (Fig. 5a).

By blue native PAGE, a Tim23–Tim17 core complex of the prese-quence translocase can be separated28 (Fig. 5b, lane 3). In contrast,Tim50, and mtHsp70 and Tim44 of the PAM, are released from thisTIM core complex and do not form defined blue native complexes, butmigrate in a broad molecular-mass range9,28,29. Pam16 and Pam18both migrated in the range of 80 kDa, distinct from the migration ofthe Tim23–Tim17 core complex (Fig. 5b), raising the possibility thatPam16 and Pam18 form a complex. To address this directly, weexpressed and purified both Pam16 and the J domain of Pam18(Pam18J). When Pam16 carrying a His-tag was immobilized on a Ni-NTA agarose column, expressed Pam18J bound to it (Fig. 5c, lane 6).

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Figure 3 pam16 mutant mitochondria aredefective in preprotein import into the matrix. (a) Serial dilutions of wild-type (WT), pam16-1and pam16-3 cells were plated on mediumcontaining either glucose or glycerol as the carbonsource, then incubated at the indicated temper-atures. (b) WT, pam16-1 and pam16-3 cells weregrown at 24 °C in YPG and subsequently shifted to37 °C for 20 h. Whole-cell extracts were analyzedby SDS-PAGE and western blotting. (c) After a15 min incubation of the mitochondria at 37 °C,radiolabeled F1β preprotein was incubated withWT, pam16-1 and pam16-3 mitochondria at 25 °Cfor the times indicated, then subjected to pro-teinase K treatment. Samples were analyzed bySDS-PAGE and digital autoradiography. Theamount of m-F1β in WT mitochondria after thelongest import time was set to 100% (control). p, precursor; m, mature. (d) Radiolabeledb2(167)∆-DHFR and b2(220)∆-DHFR preproteinswere imported into WT, pam16-1 and pam16-3mitochondria and analyzed as in c. The amount of i-b2(167)∆-DHFR or i-b2(220)∆-DHFR in WTmitochondria after the longest import time was setto 100% (control). p, precursor; i, intermediate.(e) Import into WT, pam16-1 and pam16-3mitochondria of saturating amounts of ureadenatured b2(47)-DHFR as described in c. Afterproteinase K treatment, samples were separated bySDS-PAGE and analyzed by western blotting withanti-DHFR. The amount of i-b2(47)-DHFR in WTmitochondria after the longest import time was setto 100% (control).

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This interaction seemed specific, as Pam18J bound neither to the column material itself nor to a control protein (the purified inter-membrane space domain of Tim50, which binds Tim23 (refs. 9,10))(Fig. 5c, lanes 5 and 7). We compared the binding behavior of Pam18with that of the similarly sized matrix J protein, Jac1, and found thatonly Pam18 and not Jac1 bound to purified Pam16 (Fig. 5d, lane 2).Likewise, when the expressed J domain of Pam18 was immobilized ona column, Pam16, but not Jac1, bound to it (Fig. 5d, lane 5). Together,these results show that Pam16 and Pam18 interact with each other in acomplex that migrates at ∼ 80 kDa on blue native gels.

To determine whether Pam16 is involved in the interaction ofPam18 with the TIM23 complex, we expressed Tim23 with a protein A–tag9 in pam16-3 mutant cells. Mitochondria were purifiedand then lysed with digitonin, and the presequence translocase with itsassociated components was purified by affinity chromatography.Although Tim50 and Tim44 copurified with the tagged Tim23 inpam16-3 mitochondria, the association of Pam18 with the TIM23complex was strongly reduced (Fig. 6a, lanes 7 and 8) as compared

with its association in wild-type cells expressing tagged Tim23 (Fig. 6a,lanes 5 and 6). As the levels of Pam18 in pam16-3 mitochondria wereroughly similar to that of wild-type mitochondria (Fig. 4a and Fig. 6a,lanes 1–4), a defect in Pam16 therefore impairs the association ofPam18 with the presequence translocase.

Pam18 promotes the formation of a stable complex betweenmtHsp70 and Tim44 (ref. 25). The complex formation betweenmtHsp70 and Tim44 was analyzed by coprecipitation with antibodiesdirected against either mtHsp70 or Tim44 from mitochondria lysedwith Triton X-100 at low levels of ATP19,20,25,41–43. Under both copre-cipitation conditions, the formation of a stable complex betweenmtHsp70 and Tim44 was substantially reduced in pam16 mutantmitochondria (Fig. 6b). Thus, mutations in Pam16 lead to a similardefect in the mtHsp70-Tim44 interaction, as observed for mutantPam18, indicating that both Pam proteins cooperate to promote thereaction cycle of mtHsp70.

Finally, we tested whether functional Pam16 is indeed required forPAM function at the protein import channel, that is, during transloca-

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(min)∆ψ

WTpam16-1 pam16-3

DIC2

Time (min)

c

6 121.5 3 12 6 121.5 3 12 6 121.5 3 12

+ + + – + + + – + + + –++ +

WT pam16-1 pam16-3

(min)

∆ψ

m

pi

Cyt. c1

Time (min)

Pro

cess

ed c

yt. c

1(%

of c

ontr

ol)

d

Time (min)

e

6 121.5 3 12 6 121.5 3 12 6 121.5 3 12

+ + + – + + + – + + + –++ +

WT pam16-1 pam16-3

(min)

∆ψ12 1212 12

– –+ +

WT ssc1-3

CoxVa∆

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 111213 1415 16 1718 19

1 2 3 4 5 6 7 8 9 10 1112

1 2 3 4 5 6 7 8 9 10 11 12

0 50 250Time (s)

pam16-3

0

10

20

30

Mitoch. NaCN

WT

pam16-1

f

1 2 3

WT

pam

16-1

pam

16-3

TOMcomplex

TIM23complex

TIM22complex

WT

pam

16-1

pam

16-3

WT

pam

16-1

pam

16-3

4 5 6 7 8 9

(µg) 15 30 15 30 15 30 15 30 15 30 15 30(µg)

WTpam16-1

pam16-3

0

25

50

75

100

Ass

embl

ed D

IC(%

of c

ontr

ol)

0 5 10 2015 0 5 10 150

25

50

75

100 WTpam16-1

pam16-3

Impo

rted

Cox

Va ∆

(% o

f con

trol

)

0 5 10 15

100

75

50

0

25

WT

pam16-1

pam16-3

Flu

ores

cenc

e (a

rbitr

ary

units

)

0

10

20

30

0

10

20

30

150

kDa

67

140

232

440

660

67

140

232

kDa

Figure 4 pam16 mutant mitochondria can insert preproteins into the inner membrane. (a) Mitochondrial proteins from wild-type (WT), pam16-1 and pam16-3mitochondria were separated on SDS-PAGE and analyzed by western blotting with the indicated antibodies. (b) After a 15-min, 37 °C heat shock of mitochondria,60 µg (protein) of WT, pam16-1 and pam16-3 mitochondria were analyzed by blue native PAGE and immunodecoration with antiserum against Tim23, Tom40and Tim22. (c) Membrane potential measurements in heat-treated WT, pam16-1 and pam16-3 mitochondria by fluorescence quenching. (d) Radiolabeled carrierprotein DIC was incubated at 25 °C with isolated heat-treated WT, pam16-1 and pam16-3 mitochondria for the times indicated. The samples were subsequentlytreated with proteinase K, then analyzed by blue native PAGE and digital autoradiography. The amount of DIC2 formed in WT mitochondria after the longestincubation time was set to 100% (control). (e) Radiolabeled cytochrome c1 preprotein was incubated with WT, pam16-1 and pam16-3 mitochondria asdescribed in d. After proteinase K treatment, the samples were analyzed by SDS-PAGE and digital autoradiography. The amount of processed cytochrome c1 inWT mitochondria after the longest import time was set to 100% (control). p, precursor; i, intermediate; m, mature. (f) Radiolabeled CoxVa∆26−89preprotein wasincubated with mitochondria and analyzed as in e. The amount of CoxVa∆ in WT mitochondria after the longest import time was set to 100% (control).

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tion of preproteins through the translocase channels. We used an assaythat tests the import-driving activity of the import motor on a two-membrane-spanning preprotein19,44,45. When b2(167)∆-DHFR isarrested in both the TOM and TIM23 channels in the presence ofmethotrexate, a substantial fraction of the processed preprotein is notcleaved by externally added proteinase K, although the DHFR domainis exposed on the mitochondrial surface. The inward-directed import-driving activities of the membrane potential and the mtHsp70 motorcause the folded DHFR to come so close to the outer membrane thatthe protease cannot access it, whereas upon inactivation of the mem-brane potential and mtHsp70, the preprotein slides back and the DHFRcan be accessed by proteinase K. Therefore, when the membrane poten-tial is dissipated by the ionophore valinomycin, the import-drivingactivity of mtHsp70 can be assessed by the protease resistance of theprocessed preprotein in wild-type mitochondria19,44,45 (Fig. 6c, lanes2–5, left plot). In pam16-1 and pam16-3 mitochondria, a rapid loss ofprotease resistance for i-b2(167)∆-DHFR was observed (Fig. 6c, lanes7–10 and 12–15; middle and right plots). We conclude that defects inPam16 impair the import-driving activity of the matrix import motor.

DISCUSSIONAlthough it has been assumed that the PAM of mitochondria consists of three essential components, it is now clear that two addi-tional components are essential for the function of PAM: therecently discovered Pam18 (refs. 25–27) and the newly identifiedPam16.

Pam16 is a peripheral protein of the mitochondrial inner membraneexposed to the matrix. Notably, Pam16 shows substantial similarity to Jproteins; however, it lacks the signature motif HPD of J proteins17,21–24.Consistent with this aspect, Pam16 does not stimulate the ATPase activ-ity of mtHsp70, unlike true J proteins that contain the HPD motif, suchas Pam18 (refs. 25,27). Moreover, combining Pam18 and Pam16 in theATPase assay did not stimulate the ATPase activity of Hsp70 beyond theeffect of Pam18 alone (data not shown). Pam16 is well conserved ineukaryotes, and although the function of these homologs has not beenanalyzed in great detail, it is notable that inactivation of a Drosophilamelanogaster homolog of Pam16 causes the death of larvae during earlydevelopment46. This observation demonstrates that the essential natureof Pam16 function is conserved. In addition, mammalian homologshave been localized to mitochondria and have been suggested to beinvolved in the regulation of mitochondrial activity47.

Pam16 is associated with the presequence translocase and is presentin a functional TOM–TIM–PAM supercomplex that can be isolatedwhen a preprotein is arrested in the translocase channels of both outerand inner membranes9,25,28,29. Mutant mitochondria with a defectivePam16 are selectively impaired in the translocation of preproteins intothe mitochondrial matrix and thus resemble mitochondria defectivein mtHsp70, Tim44 or Pam18, which also show defects in proteinimport (refs. 19,25–27,48). The insertion of precursor proteins intothe inner membrane by the ∆ψ-driven TIM23 complex, however, isnot impaired in pam16 mutant mitochondria.

What role does Pam16 have in the import motor? The com-position and function of the presequence translocase with theTim23–Tim17 core complex and the associated Tim50 (refs. 9–11,28,29) are not disturbed in pam16 mutant mitochondria. However,the two inner-membrane proteins, Tim44 and Pam18, that interactwith mtHsp70 are differently affected in pam16 mutants. The asso-ciation of Pam18, but not of Tim44, with the TIM23 complex isstrongly impaired when Pam16 is defective. A cooperation of Pam16and Pam18 in vivo is suggested by the finding that overexpression ofPam18, but of no other translocase or motor subunit, suppresses thetemperature-sensitive lethal growth defect of a pam16 mutant.Pam16 directly interacts with the J domain of Pam18 and may thuslink it to the TIM23 complex. It has been suggested that stimulationof the ATPase activity of mtHsp70 by Pam18 at the import channel isrequired for formation of a stable mtHsp70–Tim44 complex25–27.Pam16 is indeed required to generate a stable mtHsp70-Tim44 inter-action. These findings suggest that Pam16 may have an adapter-likefunction by forming a subcomplex with Pam18 and supporting itsassociation with the TIM23 complex. Pam16 is thereby needed togenerate an efficient import-driving activity of the mtHsp70 motorat the protein import channel.

24 °C 37 °C+ YEp352

+ PAM16

+ PAM18

+ TIM44

+ mtHSP70

+ TIM50

+ TIM23

+ TIM17

+ MDJ1

a b

440

232

140

67

kDa

1 21 2 3 4 5 6

Tim

50IM

S

Unbound Eluate

Pam18J

c

Pam16

Jac1

d

1 2 3 4 5 6

Jac1

Pam18

1 2 3 4 5 6 7

Con

trol

pam16-1

Tim

50IM

S

3

TIM23 complexPam16/Pam18

Con

trol

Load

Pam

16

Pam

16Lo

ad

Load

Ni-N

TA

Ni-N

TA+P

am16

GS

T

GS

T-P

am18

J

Pam

16P

am18

Tim

23

Figure 5 Pam16 interacts with Pam18. (a) Serial dilutions of pam16-1 cellsexpressing the indicated genes from a yeast 2µ plasmid were plated on YPDand grown at the indicated temperatures. (b) Radiolabeled Pam16, Pam18and Tim23 were imported into mitochondria. After solubilization in digitonin-containing buffer, complexes were analyzed by blue native PAGE and digitalautoradiography. (c) Immobilized His10-tagged Pam16 and His10-taggedTim50IMS (intermembrane space domain of Tim50) were incubated withpurified GST-Pam18J. Bound proteins were eluted and analyzed by SDS-PAGE and western blotting. 12% of load and unbound and 100% of eluateare shown. GST alone did not bind to Pam16 (not shown). (d) His10-taggedPam16 and Ni-NTA agarose (left) or GST-Pam18J and GST (right) wereincubated with radiolabeled Pam18 and Pam16, respectively, or with Jac1 asa control. Bound proteins were eluted and analyzed by SDS-PAGE and digitalautoradiography. 7% of load and 100% of eluate are shown.

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Pam16 was independently identified as a subunit of the mitochon-drial protein import machinery and termed Tim16 (see accompanyingpaper in this issue)49. In agreement with our analysis, the new subunitwas shown to be essential for yeast cell viability and critical for thetransport of proteins into the mitochondrial matrix.

METHODSYeast strains. Yeast strains were derived from YPH499 (ref. 50) except for PK83(ssc1-3) and the corresponding wild-type PK82 (ref. 48). Error-prone PCR wasused for the generation of temperature-conditional pam16 alleles. Gap repairand plasmid shuffling were then used to introduce the alleles into a pam16 genedeletion mutant covered with the wild-type PAM16 on a plasmid. The mutantspam16-1 (YPH-BG-mia1-1) and pam16-3 (YPH-BG-mia1-3) were selected forfurther analysis.

Isolation and analysis of mitochondria. After growth of the yeast strains ateither 30 °C or 24 °C (temperature-conditional mutants) in YPG (1% (w/v)yeast extract, 2% (w/v) peptone and 3% (v/v) glycerol) and purification asdescribed51, mitochondria were resuspended to 10 mg protein ml–1 in SEMbuffer (250 mM sucrose, 1 mM EDTA and 10 mM MOPS, pH 7.2) and stored at–80 °C. For isolation of mitochondria from pam16-3 mutant cells expressing

protein A–Tim23, cells were grown at 24 °C in selective medium with glycerolas the carbon source.

For analysis of protein localization within mitochondria, hypotonic swellingwas carried out by diluting the mitochondria in EM buffer (1 mM EDTA and10 mM MOPS, pH 7.2) to 1 mg protein ml–1 and incubating it for 10 min onice. Sonication of mitochondria (1 mg protein ml–1) was done on ice in500 mM NaCl and 10 mM Tris, pH 7.5, as described25. Subsequent treatmentby proteinase K was carried out for 15 min on ice, and 1 mM PMSF was thenadded to inhibit the protease.

To separate soluble from peripheral and integral membrane proteins, mito-chondria were either sonicated or mitochondrial membranes were extracted in100 mM Na2CO3. After centrifugation at 100,000g, the pellet and supernatantfractions were precipitated by trichloroacetic acid.

In vitro import. In vitro transcription, radiolabeling of proteins and import ofprecursor proteins into mitochondria were done as described51. Recombinantpreproteins b2(47)-DHFR and b2(167)∆-DHFR were purified and imported asdescribed25,28. Mitochondria isolated from temperature-sensitive yeast strainswere incubated at 37 °C for 15 min before import at 25 °C. For analysis of theinward-directed import activity, b2(167)∆-DHFR was imported in the presenceof 5 µM MTX and the assay was done as described19.

Protein purification and antibodies. The coding region of PAM16 (YJL104W)was amplified by PCR and cloned into pET10N for expression in Escherichiacoli. After transformation into E. coli strain BL21 and growth to an A600 of 0.5,induction was carried out with 1 mM IPTG. Cells were harvested 3 h afterinduction. After several freeze-thaw steps, the cell pellet was resuspended inbuffer A (500 mM NaCl, 1 mM PMSF, 0.1 mg ml–1 lysozyme, 0.5% (w/v)Triton X-100, 0.01 mg ml–1 DNase I, 1 mM MgCl2, and 20 mM Tris-HCl,pH 8.0) and the cells were disrupted by sonication. Extracts were clarified by12,000g centrifugation at 4 °C for 30 min. A 500 µl bed volume of Ni-NTAagarose was washed twice with buffer B (500 mM NaCl, 5 mM imidazole and20 mM Tris-HCl, pH 8.0), then incubated with cleared extracts for 1 h at 4 °C.Unbound protein was removed by washing with buffer C (500 mM NaCl,20 mM imidazole and 20 mM Tris-HCl, pH 8.0). To remove bacterial DnaK,8 mM ATP was added, and the Ni-NTA agarose was incubated at room tem-perature for 10 min and subsequently washed with buffer D (500 mM NaCl,65 mM imidazole and 20 mM Tris-HCl, pH 8.0). Bound proteins were elutedwith buffer E (500 mM NaCl, 20 mM Tris-HCl, pH 8.0 and 500 mM imida-zole), dialyzed against 30 mM Tris-HCl, pH 7.4, 100 mM KCl and 5% (v/v)glycerol, and stored at –80 °C. Cloning and purification of GST-Pam18J weredone as described25. For generation of anti-Pam16 antisera, rabbits wereinjected with denatured Pam16.

In vitro binding. His10-tagged Pam16 and His10-tagged Tim50IMS9

(intermembrane space domain of Tim50) were expressed in E. coli. 50 µl Ni-NTA agarose was incubated with cleared cell lysates and unbound proteinwas removed by extensive washing. Beads were then equilibrated in buffer

– 6 11∆t (min) 31

WT pam16-1pam16-3

– 6 1131 – 6 1131

WT pam16-1 pam16-3

0 9 1263 0 9 1263 0 9 1263Time (min)

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Pro

teas

e-re

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i-b2(

167)

∆-D

HF

R (

% o

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b2(167)∆-DHFR

c

p

i

b

a

ATP – –+ +

WT pam16-3

– –+ +

WT pam16-1

Anti-Tim44

mtHsp70

Tim44

mtHsp70

Tim44

ATP + +– –

WT pam16-1

– +

pam16-3

Copurifiedwith Tim23Load

WT pam16-3 WT pam16-3

ATP – –+ ++ +– –

Tim44

Tim50

Pam18

Tim23ProtA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8

Anti-mtHsp70

1 2 3 4 5 6 7 8

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sp70

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pam16-3

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44(%

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– + – +

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Pam

18 c

opur

ified

with

Tim

23(%

of c

ontr

ol)

Mitoch.

b2(167)∆-DHFR+MTX Valinomycin

First inc.15 min

– Prot. K

Second inc.∆t

+ Prot. K

ATP

Figure 6 Pam16 is required for the interaction of Pam18 with thepresequence translocase and for formation of a stable mtHsp70–Tim44complex. (a) Protein A–Tim23 was expressed in wild-type (WT) or pam16-3yeast cells and mitochondria were isolated. After a 15-min incubation at37 °C, mitochondria were solubilized in digitonin buffer and protein A–Tim23was purified. Bound proteins were eluted with SDS sample buffer, separatedby SDS-PAGE and analyzed by western blotting. 10% of the load and 100% ofthe eluted proteins are shown. The relative amount of Pam18 copurified withTim23 in WT mitochondria in the absence of ATP was set to 100% (control).(b) mtHsp70-Tim44 interaction was assessed by coimmunoprecipitation withantibodies against Tim44 or mtHsp70 after pretreatment of mitochondria as in a. Precipitates were analyzed as in a. The amount of mtHsp70 or Tim44,respectively, coprecipitated in WT mitochondria in the absence of ATP was set to 100% (control). (c) For analysis of the inward-directed import-drivingactivity, mitochondria were pretreated as in a, and then incubated at 25 °Cwith b2(167)∆-DHFR in the presence of MTX. After dissipation of themembrane potential, samples were either left at 25 °C for ∆t, then treated with proteinase K, or left untreated. Samples were analyzed by SDS-PAGE and digital autoradiography. The amount of i-b2(167)∆-DHFR in non-protease-treated samples was set to 100% (control). p, precursor; i, intermediate.

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PDC (20 mM HEPES-KOH, pH 7.5, 100 mM KOAc, 10 mM Mg(OAc)2, 10% (v/v) glycerol, 20 mM imidazole, and 0.5% (w/v) Triton X-100). Purified GST-Pam18J was diluted in buffer PDC and 2 mM PMSF,added to the preloaded Ni-NTA agarose and incubated for 1 h at 4 °C. Beadswere washed 10× in buffer PDD (20 mM HEPES-KOH, pH 7.5, 100 mMKOAc, 10 mM Mg(OAc)2, 10% (v/v) glycerol, 20 mM imidazole, and 0.25%(w/v) Triton X-100). Bound protein was eluted with buffer E, separated onSDS-PAGE and analyzed by western blotting.

Purified His10-tagged Pam16 and GST-Pam18J were bound to Ni-NTAagarose or glutathione-Sepharose, respectively. Beads were washed and equili-brated in buffer PDC. Protease inhibitors (Complete EDTA free, Roche) wereadded and the beads were incubated together with in vitro–synthesized 35S-labeled proteins for 1 h at 4 °C. Beads were washed 10× with buffer PDDand bound protein was eluted in two steps, first with 20 µl buffer E and subse-quently with 20 µl 2× Laemmli sample buffer. The two eluates were combined,separated on SDS-PAGE and then analyzed by digital autoradiography.

Miscellaneous. After protein separation on PAGE and transfer to PVDF mem-brane, western blotting was done according to standard techniques and proteinswere detected by enhanced chemiluminescence (Amersham). Membrane poten-tial measurements were carried out as described14. For some gels, irrelevantlanes were digitally excised. Isolation of the TIM23 complex via proteinA–Tim23, generation and isolation of a TOM-TIM translocation intermediate,in-gel protein digests, and preparation of nano-HPLC and ESI mass spectro-metry were carried out as described9. Coimmunoprecipitations were carried outas described19. Blue native PAGE analysis was done essentially as described28.

ACKNOWLEDGMENTSWe thank R. Jensen for antiserum against Tim17, S. Rospert for antiserum against Pam18 and I. Perschil, N. Zufall and H. Müller for expert technicalassistance. This work was supported by the Deutsche Forschungsgemeinschaft,Sonderforschungsbereich 388, Max Planck Research Award, Bundesministeriumfür Bildung und Forschung, Nationales Genomforschungsnetz, and the Fonds derChemischen Industrie. M.L. is a recipient of a postdoctoral fellowship from theWenner-Gren foundations. Work in the laboratory of M.G.C. was supported bygrant GM 57017 from the US National Institutes of Health.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 8 December 2003; accepted 23 January 2004Published online at http://www.nature.com/natstructmolbiol/

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