Mitochondrial Import of the ADP/ATP Carrier Protein in ...

Vol. 263. No. 14,Issue of May 15, pp. 6783-6790, 1988 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry nnd Molecular Biology, Inc

Mitochondrial Import of the ADP/ATP Carrier Protein in Saccharomyces cerevisiae SEQUENCES REQUIRED FOR RECEPTORBINDING

AND MEMBRANE TRANSLOCATION* (Received for publication, October 30, 1987)

Cynthia Smagula andMichael G.Douglas$ From the Department of Biochemistry, Southwestern Graduate Schnot of Biomedical Sciences, University of Texas Health Science Center at Dallas, Dallas, Texas 75235

The ADP/ATP carrier of yeast (309amino acids) is in some detail using specific inhibitors of the translocator an abundant transmembrane proteinof the mitochon- which are able to interact specifically with the protein from drial inner membrane whose import involves well- either the mitochondrial matrix or cytoplasmic face of the defined steps (Pfanner,N., and Neupert, W. (1987)J. transmembrane structure. Analysis of the primary sequence Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADPfATP of the translocator from a variety of sources indicates that carrier (AAC) sequences at the amino terminus and the primary structure of this membrane-spanning transporter mouse dihydrofolate reductase(DHFR) at the carboxyl is highly conserved (Adrian et al., 1986). Recent analysis of terminus indicates that the first72 amino acids of the different solute transporters of the mitochondrial inner memsoluble carrier protein, a hydrophilic region of the brane indicate that theADP/ATP carrier is representative of protein, are not by themselves sufficient for initial a family of integral proteins in this membrane which share binding to theAAC receptor on the mitochondrial sur- considerable sequence homology and membrane topology face. However, an AAC-DHFR gene fusion containing (Aquila et al., 1985; Runswick, et al., 1987). It is possible that the first 11 1 residues of the ADP/ATP carrier protein these structurally related proteins may share acommon route exhibited bindingto mitochondria at low temperature of import and assembly in the inner membrane. (2 “C) and internalization at 25 “C to a mitochondrial space protected from proteinaseK in the same manner The ADP/ATP carrier protein is the most abundant protein as the wild-type ADP/ATP carrier protein. The AAC- in mitochondria from different sources, constituting 14-20% DHFR protein, in contrast to the wild-type AAC pro- of the total mitochondrial membrane protein (Klingenberg, tein imported into mitochondria under optimal condi- 1985). Its biogenesis has been examined in detail using in uivo tions, remained extractable at alkaIine pH and ap- and in vitro techniques. Unlike the mitochondrial precursors peared to be blocked at an intermediate step in the which contain transient presequences for localization in miAAC import pathway. Based on its extraction proper- tochondria, the ADP/ATP carrier protein is imported into ties, this AAC-DHFR hybrid is proposed to be associ- the organelle inner membrane without any apparent proteoated with a proteinaceous component of the import apparatus within mitochondria. These data indicate lytic processing (Pfanner and Neupert, 1987a). Early studies that the import determinants for theAAC protein are revealed that thebinding of the soluble AAC protein prepared in a cell-free translation lysate was mediated by a proteasenot located at its extreme amino terminus and that 11 1 residues of sensitive component which appeared to be distinct from that protein determinants distal to the first the carrier may be necessary to move the protein be- required for the efficient import of a presequence-containing yond the alkali-extractable stepin the biogenesis of a mitochondrial precursor (Zwizinkski et al., 1984).These studfunctional AAC protein. ies and more recent analyses have clearly defined the mitochondrial apparatus required for binding and initial translocation of the AAC protein into mitochondria (Schwaiger et Transport of adenine nucleotides between mitochondrial al., 1987). This receptor appears to be distinct from that and cytoplasmic compartments of the cell is mediated by an mediating the binding of the F,-ATPase 6-subunitprecursor. integral homodimeric proteinin the mitochondrial inner These studies demonstratedthat binding of the soluble AAC membrane called the adenine nucleotide translocator or ADP/ protein required a trypsin-sensitive component whichwas ATP carrier protein(Klingenberg, 1981). The transport propeasily removed from mitochondria. Under conditions which erties of the ADP/ATP carrier (AAC)’ have been examined have been documented to solubilize selectively the mitochon* This work wassupported inpart by National Institutes of Health drial outer membrane (Schwaiger et al., 1987), the release of Grants GM26713 and GM36537. The costs of publication of this the ADP/ATP carrier precursor paralleled the release of the article were defrayed in part by the payment of page charges. This outer membrane protein porin. article must therefore be hereby marked “advertisement” in accordMitochondrial binding and insertion of the AAC protein ance with 18 U.S.C. Section 1734 solely to indicate this fact. into the mitochondrial inner membrane have been resolved $ Recipient of Grant 1-814 from The Robert A. Welch Foundation. Submitted this work in partial fulfillment of the requirements for a into five distinct steps based on the combination of protease Ph.D. degree. T o whom reprint requests should be addressed Dept. protection, alkali extraction, temperature shift, and chromatof Biochemistry, University of Texas Health Science Center a t Dallas, ographic studies (Pfanner and Neupert, 1987b). The soluble 5323 Harry Hines Blvd., Dallas, T X 75235. ADP/ATP carrier protein (stage 1) can bind to themitochonThe abbreviations used are: AAC, ADP/ATP carrier; DHFR, drial outer membrane (stage 2) in the absence of a membrane dihydrofolate reductase; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid SDS, sodium dodecyl sulfate, MOPS, 3-(N- potential to a protease-sensitive component. Following this morpho1ino)propanesulfonicacid; PMSF, phenylmethylsulfonyl flu- binding, the AAC protein is sequestered in some manner oride. (stage 3) such that it is no longer accessible to proteinases ~~~~~~

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6783

6784

ADPIATP Carrier Protein Import

outside mitochondria. The stage 3 translocational intermediate is not fixed intoa transmembrane complex in the bilayer because i t can be solubilized by alkali extraction. E n t r y or association of the translocational intermediate presumably into regions ofcontact between the outer and inner membrane (stage 4) is followed by assembly as the dimer into the inner membrane (stage 5). This last step is characterized by an alkaliextraction-resistantform of the AAC protein. The presence of stage 4 and 5 forms of the ADP/ATP carrier i n mitochondria is increased in the presence of a membrane potential across the mitochondrialinnermembrane. This observation has led to the model that the membrane potential is required for the later assembly steps in the translocator import pathway. At this time,there are no data on the sequences ofthe AAC protein which are involved in a n y step of t h i s import sequence, and i t is not known if different regions of the translocator participate in different steps. T h e AAC protein in yeast which is 309 amino acids in length is homologous to solute carrier proteinsfromothermitochondrialsources(Adrian et al., 1986). Earlier genefusion studies have revealed that the first 115 residues of the AAC protein are sufficient to target /3galactosidase fused to i t to mitochondria in vivo (Adrian et al., 1986). In order to examine directly the role of the first 115 residues in mitochondrial import, we have characterized the behavior of gene fusions between different amino-terminal lengths of the AAC protein i n this region and a soluble reporter protein mouse dihydrofolate reductase. DHFR is a soluble cytoplasmic protein whichdoes not bind to mitochondria unless it contains the appropriate mitochondrial import signals at its amino terminus (Hurtet al., 1984, 1985). This study demonstratesthat, unlike the basic hydrophilic mitochondrial import signals of presequences which are located at the extreme amino terminus, the import signal for the AAC protein is within a region which begins 72 residues from the amino terminus of the protein. In addition, this analysis indicates that the first 111 residues consisting of a putativemembrane domain (residues 72-97) and a highly charged region (residues 97-111) are sufficient to deliver the protein only to an intermediate stage (stage 3) inthe import pathway of the AAC protein. This stage 3 intermediate is found even in the presence of an energized membrane, contransport of the complete AAC protein ditions which promote to the inner membrane. Thus,the import of the AAC protein to its final destination in the mitochondrial inner membrane may involve protein determinantsdistal t o t h e first 111residues of the protein. EXPERIMENTAL PROCEDURES

Strains and Medin-Mitochondria were isolated from Saccharomyces cereukiue strain D273-10B (MATor) grown in semisynthetic salts, 2% lactate medium (Daum et al., 1982) to an optical density (600 nm) of 1.0-1.2. Yield was about 5 g of cells/liter. Escherichia coli MC1066 (F- A.IacX74 gam gam rpsL hsdR trpC9830 leuB600 pyrF74:Tn5) (Casadaban and Cohen, 1980) was used for amplification of plasmids. Ampicillin was added to media to select ampicillinresistant transformants (Maniatis et al., 1982). E. coli JMlOl (F- lac pro supE traD36) was used for M13 sequencing. DNA Methods and Construction of Plasmids-The HindIII-Sal1 fragment of pFDll (Simonsen and Levinson, 1983), containing 187 codons of mouse dihydrofolate reductase DNA, was inserted into the multilinker region of pT7-2 (Genescribe). pT7-2 is a transcription vector that places the T, polymerase promoter adjacent to the multilinker region. An EcoRI-SstI fragment derived from TZ115 (Adrian et ai., 1986) containing the first 115condons of the ADP/ATP carrier plus 2 kilobases of the lOcZ gene was inserted into the EcoRI-Sa01 sites of pUC19 (International Biotechnologies, Inc.). The pT7-2 DHFR vector was opened at theunique HindIII site,and theHindIII fragment of pUC19:TZllB was inserted (Fig. 1).The construct thus

generated, pTZD, was linearized a t the unique BamHI site located a t the junction of the ADP/ATP carrier and lacZ DNA. Limited Bal31 digestion was followed by restriction at PstI, filling in with T, DNA polymerase and ligation with T4 DNA ligase, generating the pTD vector series. The HindIII fragments of the pTD vectors were sized on a 1.5% agarose gel. The pTDplasmid was linearized by digestion at Sal1 and transcribed with T7polymerase (Chen and Douglas, 1987). Transcripts were translated in reticulocyte lysate (Promega Biotec) in the presence of [36S]methionine(Du Pont-New England Nuclear). Those pTD vectors that translated a protein larger than DHFR were sequenced by the dideoxy method (Sanger et al., 1980). Translation reactions to be used for in vitro import were frozen in liquid nitrogen and stored at -70 "C. DNA techniques were as described by manufacturers or according to Maniatis et al. (1982). ATP1 (Takeda et al., 1986) and full-length AACl (Adrian et al., 1986) were inpT7-2 transcription vectors. The vectors were linearized at a BamHI site in the multilinker, transcribed, and translated as described above. Mitochondrial Subfractwnutwn-Mitochondria were isolated (Daum et al., 1982) and suspended in 0.4 ml of 0.6 M mannitol, 10 mM Tris-C1 (pH 7.4). The import reaction consisted of 200 pl of mitochondria (1-2 mg of total protein) and 75 pl each of reticulocyte lysate containing 35S-labeledproteins. Final volume wa8 1.0 ml. The import reaction (Gasser et al., 1982) included 0.6 M mannitol, 20 mM HEPES/KOH (pH 7.4), 1 mM ATP, 1 mM MgCl,, 5 mM phosphoenolpyruvate, 40 units of pyruvate kinase, 1 mM dithiothreitol. After incubation at 30 "C for 30 min, the import mixture was treated with 250 pg/ml proteinase K (Sigma, Type XI) at 0 "C for 30 min. PMSF was added to 1 mM, and mitochondria were reisolated through a sucrose cushion (0.6 ml of 20% sucrose in 10 mM Tris-HC1 (pH 7.4). The samples were centrifuged in a Beckman Microfuge a t 13,000 X g for 10 min. The pellet was resuspended in 80 pl of 0.6 M mannitol/ HEPES (pH 7.4) to which 400 pl of cold 10 mM Tris-C1 (pH 8.0), 1 mM PMSF was added. Mitochondria were incubated for 10 min at 0 "C and reisolated by centrifugation at 13,000 X,g for 10 min. The supernatant contained proteins of the intermembrane space. The pellet was resuspended in 0.6 M sucrose, 10 mM Tris (pH 7.4), 3 mM MgCI,, 3 mM ATP, 1 mM PMSF and sonicated twice for 60 s each with a 60-s interval using a Branson sonifier (at setting 1,0.1% duty). Membranes were separated from soluble proteins a t 107,000 X.,g for 20 min a t 4 "C. Aliquots from each stage of the subfractionation procedure were suspended in 4 X SDS sample buffer and analyzed by gel electrophoresis using a 12.5% acrylamide-SDS gel (Laemmli, 1970). After electrophoresis, the gelwas treated for fluorography using sodium salicylate (Chamberlain, 1979).The gel wasdried down, and a fluorograph was made at -70 "C using Kodak A-AR film. Quantitation of the relative amounts of labeled protein present within an experiment was determined by scanning of fluorograms ofgel following different times of exposure with a scanning densitometer (E-C Apparatus Corp.) and quantitating the relative areas under the peaks. Binding and Import of Labeled Proteins-Mitochondria were isolated (Daum et al., 1982) and resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS/KOH (pH 7.2) (Pfanner andNeupert, 198%). The import reaction consisted of 3.5-7 pl of reticulocyte lysate and 20 pI(10-20 pg) of mitochondria/100 pl of total volume. The binding and import reactions contained, in addition, a buffer consisting of 250 mM sucrose, 10 mM MOPS/KOH (pH 7.21, 80 mM KCl, 5 mM MgCl,, and 3% bovine serum albumin (Pfanner andNeupert, 1987b). Inhibitors of membrane potential, valinomycin and oligomycin (both from Sigma), were added from 100 X ethanol solutions where indicated. The import reactions were incubated at 25 "C for 25 min and then divided into equal aliquots. The reactions were treated with 1 mM PMSF or 25 pg/ml proteinase K for 30 min on ice. PMSF (1 mM) was then added to the proteinase K-digested reactions, and incubation on ice continued for an additional 5 min. Mitochondria were reisolated through 0.6mlof 20% sucrose, 10 mM Tris-C1 (pH for 10 min. The pellet was resuspended in 4 X 7.4) at 13,000 x gmax SDS sample buffer and analyzed as described above. Alkaline Extraction of Protease-treated Mitochondria-In order to assess localization of hybrid proteins to soluble or membrane compartments, mitochondria were incubated with 35S-labeled proteins and treated with proteinase K as described above. Following incubations with PMSF, the mitochondria were then reisolated through sucrose, resuspended in 0.1 M Na2C03 (pH11.5) (Fujiki et al., 1982a, 1982b) at a concentration of 100-200 pg/100 p1, and incubated for 30 for 1 h. min on ice. Membranes were sedimented at 257,000 X gmax The supernatantwas neutralized, and all samples were suspended in

ADPIATPImport Carrier Protein 4 X SDS sample buffer for gel analysis. A fluorograph was made of the dried gel as described above. RESULTS

Gene Fusions to AACl -The gene encoding the yeast ADP/ ATP carrier proteinwas isolated by genetic complementation of the PET9 or OP1 mutation in earlier studies(O’Malley et al., 1982). Characterization of this gene has shown that it exhibits >70% identity with AAC proteins characterizedfrom different organisms (Adrian et al., 1986). In this study, we have renamed this gene AACl to designate it by more conventional nomenclature as the gene encoding the ADP/ATP carrier protein.Expression of AACl-lucZ genefusions in yeast revealed in earlier work that the first 115 residues of the ADP/ATP carrier were sufficient to direct the hybrid protein to mitochondria (Adrian et al., 1986). In order to examine the role of these amino-terminal residues in further detail, selected gene fusions encoding various amino-terminal lengths of this region fused to mouse dihydrofolate reductase were constructed (Fig. 1) for analysis in an in vitro import assay (see “Experimental Procedures”). The recipient transcription vector containing the mouse DHFR genewas constructed by ligating a 1.2-kilobase HindIII-SalI fragment from plasmid pFDll (Simonsen and Levinson, 1983) adjacent to thebacteriophage T7promoter in plasmid pT7-2. This transcriptionplasmid places the HindIII site, which is 27 base pairs upstream of the DHFR transla-

6785

tional start, adjacent to the T7promoter. Transcription and translation of this vector yield a full-length DHFR translation product. This vector also serves as a recipient for a 0.5kilobase HindIII fragment containing 115 codons of AACl fused to 641 condons of E. coli lacZ.This AACl -lucZ fragment was derived from plasmid TZ115 described previously (Adrian et al., 1986). The resulting plasmid, pTZD, contains 115 codons of AACl-coding sequence proximal to theTTpromoter and separated from DHFR sequenced by 2 kilobases of lac2 DNA (see Fig. 1).Digestion of BarnHI-restricted DNA with the processive exonuclease Ba131 was followed by Pst digestion and fill-in. This resulted in a family of gene fusions between AACl and DHFR which retained a HindIIIsite adjacent to thefusion joint (see “Experimental Procedures”). Gene fusions between AACl and DHFR were screened for continuous reading frame using transcription/translation assays as well as DNA sequence analysis. In thisstudy, three of many AACl -DHFR fusions characterized in this manner were isolated for further study.Fig. 2 depicts the fusions harboring 21, 72, and 111 codons of AAC fused to DHFR. In each case, a linker region encoding the sequence XHASLSILEFAI is present in the fusion protein. The translation products of these gene fusions translated in reticulocyte lysate were 24, 29, and 32 kDa for the 21AAC-DHFR, 72AAC-DHFR, and 111AAC-DHFR forms of the hybrid proteins, respectively. Since each of these constructions retainedthe initiator AUG EcoRI

Sac1 K p n I BarnH*I

PSI1 Hrndm

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FIG. 1. Construction of pTD transcription vector series used for in vitro import studies. Details of the TZ115 construct containingthe first115 amino acids of the yeast ADPIATP carrier and all of the E. coli IOCZ gene except the first eightcodons are published (Adrian et al., 1986). The vector containing full-length mouse DHFR, pFD11, is as described (Simonsen and Levinson, 1983). The pT7-based constructs all retained the T7 RNA polymerase promoter. The EcoRI-SstI fragment of TZ115 was inserted into pUC19, and the HindIII-SalI fragment of pFDll was moved into thepT7-2. Subsequently, the HindIII fragment of pUC19TZ115 was moved intothe Hind111 site of pT72:DHFR. Linearization of the pTZD vector thus generated at the BarnHI site was followed by limited Ea131 digestion and release of IOCZ DNA a t the PstIsite. Following T, DNA polymerase fill-in of ends, the vector was ligated to form the pTD vector series, encoding various lengths of A A C l upstream of DHFR. Constructs in which the start codon of AAC is in-frame with the startcodon of DHFR were analyzed by transcription1 translation, followed by DNA sequence analysis. k b , kilobase.

BomHI{ - 4 . 0 hb

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pUC49: T I I I Z 7.1 hb

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ImportADPIATP Protein Carrier

6786

25 'C for 30 min (see "Experimental Procedures"). Following incubation, mitochondria were separated from the reticulocyte lysate reaction mixture by sedimentation through a suAAC N P c -34 crose cushion. Shown in Fig. 3 is an autoradiogram of mito21 chondrial pellet fractions for each construct following SDS DHFR 21AACI 24 DHFR I C gel separation. The 111AAC-DHFR protein exhibited both binding and import to thesame extent observed for the AAC 72 DHFR 720$8- N C 29 protein alone (see below). However, under these conditions, less than 2% binding of the input protein could be observed 111 DHFR for either the 21AAC-DHFR or 72AAC-DHFR protein (see C 32 "Experimental Procedures"). In each case, the bound 21AACFIG. 2. 2IAAC-DHFR, 72AAC-DHFR, and 111AAC-DHFR fusion proteins. The locations of predicted transmembrane hydro- DHFR and 72AAC-DHFR proteins remained completely acphobic domains within the full-length ADP/ATP carrier protein of cessible to added proteinase K. DHFR alone under these S. cereuisiae are indicated atthe top (hatched boxes). The fusion conditions exhibited no detectable binding to mitochondria. junction, consisting of the 12-amino acid sequence XHASLSILEFAI, In separate experiments, even under conditions in which the is designated by a squiggle for each of the AAC-DHFR constructs. shorter forms of the hybrid protein were incubated in large The number ofpositively and negatively charged residues is indicated for the hydrophilic domains of the AAC protein in each of the excess over those used in Fig. 3, no significant binding could constructs. An estimate of the molecular mass of each protein is given be observed. The ATPl gene product under these conditions exhibited at the right. import to a protease-protected location and processing by the matrix metalloprotease (see below) as previously described mito mito mlto mito r. I. r.I.-r.l. r.1. (Takeda et dl., 1986). The complete lack of binding observed pK. - + " for the shorter forms of the AAC-DHFR proteins indicated .c. I11AACthat the binding observed for the 111AAC-DHFR protein DHFR ' 1 represented a specific association with components on the 72AAC-/ mitochondrial membrane. Earlier studies indicated that proDHFR / teinaceous components on the surface mediate binding of the 21AACm DHFR AAC protein (Zwizinski et al., 1984; Schwaiger et al., 1987). Binding and Import of 111AAC-DHFR in MitochondriuDHFR (I To define further theinteraction of 111AAC-DHFRwith the mitochondrial surface and itsrelationship to theAAC import A B C D E F G H I J K L pathway, we took advantage of the observation that binding FIG. 3. Bindingand import of 2XAAC-DHFR, 72AAC- of the ADP/ATP carrier protein to mitochondria at 2 "C DHFR, and 11 1AAC-DHFR proteins andDHFR to mitochonprevented its movement from stage 2 to stage 3. The bound dria. Reticulocyte lysate containing "S-labeled proteins was incubated with mitochondria in a buffer containing 250 mM sucrose, 80 AAC protein is not moved from the mitochondrial surface to mM KCI, 5 mM MgCI,, 10 mM MOPS/KOH (pH 7.2) for 25 min at some internal location and therefore remains accessible to 25 "C. The reactions were divided and treated with 1 mM PMSF or protease (Pfanner and Neupert, 1987b). As shown in Fig. 4, 25 pg/ml proteinase K (P.K.) for 30 min. PMSF (1 mM) was added when the 111AAC-DHFR protein is bound to mitochondria to protease-treated reactions, and incubation on ice was continued at 2 "C, it remains accessible to subsequently added proteinase for 5 min. Mitochondria were reisolated through sucrose, and the pellet was analyzed by gel electrophoresis. Lanes A, D,G, and J,40% K. Essentially, the same protease accessibility is observed for of the reticulocyte lysate (r.l.) incubated with mitochondria (mito) the bound AAC protein under these conditions (Fig. 4, lane analyzed in adjacent lanes; lanes B, E, H,and K , binding of proteins C). As a control, the ATPl gene product whichwas also to reisolated mitochondria not treated with proteinase K lanes C, F, included in the incubation with AAC exhibited a translocaI, and L,import of proteins to a protease-protected location within tional intermediate form of the mature F1-ATPase a-subunit mitochondria. DHFR present in 111AAC-DHFR reticulocyte lysate (lane G, lower band) is translated from the DHFR start codon which a t 2 "C. In this case, the mature protein spanning both the inner and outer membranes remained accessible to proteinase is retained in the construct. K (Fig. 4, lane C). The incubations performed here were not of the dihydrofolate reductase, internal initiation yielded vari- optimal for ATPl import since the only ATP present in the able amounts of the complete DHFR protein (Fig. 3, lanes A, import incubation was that added with the translation lysate. D,and G ) inaddition to the AAC-DHFR fusion product. However, at 2 "C, only 7% of the input ATP gene product Ligation of a HindIII-BglII fragment encoding the full-length was bound to mitochondria. Upon a shift to25 "C, there was ADP/ATP carrier protein directly into pT7-2 yielded the full- increased F,-ATPase a-subunit precursor binding; however, length AAC gene product (M, 34,000). For these studies, the in the presence of valinomycin to block maintenance of a ATPl gene encoding the F,-ATPase a-subunit precursor membrane potential prior to the shift from 2 to 25 "C, very served as acontrol to establishdistinctions between the little bound a-subunit exhibited protease protection (Fig. 4, import of a presequence-containing protein destined for the lane E). However, at thistemperature and in the absence of mitochondrial matrix and that for transmembrane insertion a membrane potential, both the 111AAC-DHFR and AAC at the inner membrane (Takeda et al., 1986). The precursor proteins exhibited efficient internalization to a protease-proof the F,-ATPase a-subunit is a protein 61kDa in size which tected space (Fig. 4, lane E). 111AAC-DHFR Is Blocked at an Intermediate Stage of is processed to a mature protein of 58 kDa by a metalloproImport-It was noteworthy that essentially all the bound AAC tease in themitochondrial matrix (Takeda etal., 1986). Binding of the AAC Protein to Energized Mitochondria and 111AAC-DHFR proteins were efficiently internalized even under conditions in which the sole source of ATP for Requires Residues beyond the First 72 Amino Acids-AACDHFR gene fusion products containing different amino-ter- energizing mitochondria was relatively low (-5 PM ATP). In minal lengths of the AAC protein were incubated with isolated a previous study (Pfanner andNeupert, 1987b), it was shown mitochondria in an in uitro mitochondrial import reaction a t that efficient localization of the AAC protein toa space KDo(oPP)

71 97 114 119

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FIG.4. Protease accessibility of ATPI, full-length AAC,

and 111AAC-DHFR proteins following binding at 2 OC and shift to 25 OC. Mitochondria were incubated in reticulocyte lysate in a buffer containing 250 mM sucrose, 80 mM KCI, 5 mM MgCI,, and 10 mM MOPS/KOH (pH 7.2) for 30 min on ice. One aliquot was removed, 1 mM PMSF was added, and mitochondria were reisolated (lanes B ) . To a second aliquot, 25 pg/ml proteinase K was added, incubation on ice wascontinued 30 min, 1mM PMSF was added, and mitochondria were reisolated (lanes C). To a third aliquot, 1 p~ valinomycin was added, and the mixture was incubated a t 25 "C for 30 min. After cooling on ice for 5 min, the reaction was divided, 1 mM PMSF was added to half, and mitochondria were reisolated (lanes D).To the other half, 25 pg/ml proteinase K was added, incubation on ice was continued for 30 min, 1 p~ PMSF was added, and mitochondria were reisolated (lanes E ) . Lunes A represent 20%of reticulocyte lysate used for incubation with mitochondria; p , FIATPase precursor form; rn, FIATPase mature form.

A B C D E F G

FIG.5. Alkaline extraction of mitochondria following incubation with A T P l , AAC, and 111AAC-DHFR proteins. Mitochondria in buffer containing 3% bovine serum albumin were pretreated with 1 p M valinomycin (Val.) and 20 p~ oligomycin (Oligo.) (lanes B ) or an equivalent volume of ethanol (lanes E ) for 5 min a t 25 "C.Reticulocyte lysate containing 35S-labeledproteins ( l a n e A ) were added, and theincubation was continued for 25 min a t 25 'C. The mixture wascooled to 0 "C, and 25 pg/ml proteinase K was added. After 30 min on ice, 1 mM PMSF was added, mitochondria were isolated (lanes B and E ) , resuspended in 0.2 M Na2CO3(pH 11.5), incubated 30 min a t 0 "C,and soluble proteins were separated from integral membrane proteins by centrifugation. Lanes C and F, supernatant following centrifugation; lanes D and G, membranes.

pellet (lanes D and G) and supernatant (lanes C and F).In de-energized mitochondria (lanes B-D), the F1-ATPase asubunit is not observed with the protease-treated mitochondria. This is anticipated since the membrane potential is required to drive import of a presequence-containingprecursor such as the F1-ATPase a-subunit precursor into a proprotected from protease (stage 3) occurred in the absence of tease-protected locationin the organelle(seealsoFig. 4). a membrane potential. However, assemblyof the protein into Furthermore, binding of presequence-containing mitochona transmembrane-bound form could occur only under condi- drial precursors to mitochondria in the absence of an enertions in which the mitochondrial inner membrane was ener- gized inner membrane isvery low or, in some cases, doesnot gized. In this study, the ability to distinguish AAC protein in occur at all (Pfanner et al., 1987b). On the other hand, both a transmembrane-bound form uersus a more peripheral asso- the AAC and 111AAC-DHFR proteins exhibitedefficient ciation with the membrane in a protease-protected space took import into a protease-protected space of the de-energized advantage of an alkaline extraction protocolforresolving organelle (laneB ) . In fact, the level of import of the two AAC peripheral and integral membrane proteins (Pfanner et al., proteins was essentially the same as that observed in the 1987b).It has been shownthat treatment of membranes with absence of inhibitors. Under these latter conditions of an 100 mM sodium carbonate will release solubleand peripheral energized membrane, efficient import and protection of the membrane proteins from the bilayer, but not integral mem- F1-ATPase a-subunit were readily apparent (lane E). brane proteins which remain pelletable with the membrane When the protected forms of the proteins were characterfraction (Fujiki et al., 1982a, 1982b). In this study, we used ized for their solubility upon alkali extraction, we observed the Fl-ATPase a-subunit as a marker for a peripheral mem- that approximately 50% of the AAC protein was extractable brane protein. When this subunit is either assembled with in de-energized mitochondria (Fig. 5, lane C) or in energized other subunits into the F1-ATPase or associated with mem- mitochondria (lane F). Full-length AAC proteins remained brane in the unassembled state, it can be released in soluble entirely with the mitochondrial membrane pellet fromenerformfrom the organellemembrane by sonication or salt gized mitochondria (lane G). These extraction conditions treatment (Todd and Douglas, 1981). quantitatively solubilized the peripherallyassociated F1As shown in Fig. 5, mitochondria which were either un- ATPase a-subunit (lane F). The extractability of the AAC treated or de-energizedwith 20 p M oligomycin plus 1 PM protein withsodium carbonate following import into devalinomycin were mixed with reticulocyte lysate containing energizedmitochondria was qualitativelyidentical to that either the F1-ATPase a-subunit precursor and AAC protein reported earlier (Pfanner and Neupert, 1987b). These obseror the 111AAC-DHFRprotein. Following the import reaction, vations led to the proposal that an energized membrane was mitochondria were treated with proteinase K and then pel- required to drive the AAC protein to its alkali-nonextractable leted through a sucrose cushion. In each case, half of the transmembrane-associatedform. It has been further proposed mitochondrial pellet was taken directly for gel analysis (lanes that the alkali-extractable translocational intermediate form B and E ) , and the other half was extracted with 100 mM of the AAC protein represented its association witha proteinsodium carbonate and then resolved by centrifugation into a aceous component either on or within the mitochondrial

6788

ImportADPIATP Protein Carrier

membranes(Pfanner et al., 1988). Theprotease-protected Takentogether with the solubility properties discussed 111AAC-DHFR protein, however, was readily extractable to above (Fig. 5), the 111AAC-DHFR protein appears to reprethe same extent by sodium carbonate from either energized sent a translocational intermediate (stage 3) in the proposed or de-energized mitochondria (comparelanes C and F ) . Thus, import pathway for the AAC protein (Pfanner and Neupert, the presenceof an energized inner membrane which dramat- 1987b). This intermediate is associated with mitochondrial ically influenced import of the F,-ATPase a-subunit precursormembranes inaspace protected from protease, but in an alkali-extractable form. In the case of the 111AAC-DHFR and transmembrane insertion of the AAC protein into the membrane had no discernibleeffect on thelocalization of the protein, its association either peripherally or integrally with some proteinaceous components of the membrane does not 111AAC-DHFR protein. 11IAAC-DHFR Translocational Intermediate Is Membrane- appearto be influenced by a membrane potential. The bound-A mitochondrialsubfractionationexperiment was 111AAC-DHFR protein appears to have reached a terminal to further due either performed to determine if the 111AAC-DHFR protein was position a t stage 3 and is unable proceed either localized as a soluble translocation intermediate within to the absence of sequences distal to residue 111or to interone of the twosoluble compartments of mitochondria or was ference of the DHFR moiety with determinants in the first associated with the membrane. Following incubation of the 111 residues. In either case, however, the data are consistent ATPI, AACI, and 111AAC-DHFR gene products with mito- with the possibility that movement of the AAC protein chondria, the incubation mixture was treated with proteinase through different stages of the import pathway may require K and separated into mitochondrial and supernatant frac- the participationof different domainsof the protein. Studies in tions. Subfractionation of the proteinase K-treated mitochon-to examine this are currently progress. dria into the intermembrane space, membrane, and matrix DISCUSSION fractions followed (Fig. 6). Throughout the subfractionation, Recent analysisof the protein signalswhich direct protein the 111AAC-DHFR protein cofractionated with the membrane fraction in the same manner as the full-length AACl localization into mitochondria has focused on the transient protein (lanes D, F, and H ) even under conditions which sequences which specify delivery to the matrix or the interutilized relatively high ionic strength. In addition, thiscosed- membrane space (for review, see Douglas et al., 1986). The imentation with membranes occurredeven under conditions matrix delivery signals are hydrophilic sequences capable of of relatively harsh hypotonic treatment release to components generatingstructureswith a highhydrophobicmovement (Von Heijne, 1986; Roise et al., 1986). These protein elements of the intermembranespace. In this experiment, the hypotonic lysis conditions also released soluble mature F,-ATPase a- at the amino terminus of a protein have been shown to be subunit (lane E ) , which has previously been shown to reside necessary and sufficient to catalyze the entry of any protein membranes of the in the mitochondrial matrix.Although these observations do fused carboxyl-terminal to it through both not define which of the two membranes the111AAC-DHFR mitochondria. The most recent analysisof the matrixdelivery pathway suggests that this transmembrane delivery occurs protein is localized to, they do show that this internalized through a hydrophilic membrane environment (Pfanneret al., intermediate remains firmly associated with the membrane 1987b). Addition of a n appropriate second localization signal, fraction in a protected space. usually a transmembrane-spanning sequence, adjacent to the matrix targeting signal serves to localize further the protein either to the outer membrane (Hase et al., 1984) or to the intermembrane space (Van Loon et al., 1986; Van Loon and Schatz, 1987; Hartl et al., 1986). These hydrophobic secondary localization signals act to direct export of an intermediate form of the protein from the mitochondrial matrix (Hartl et al., 1986). With theexception of the special case of cytochrome c, the protein elements characterized to date which direct protein AAC localization to sites within mitochondria beyond the outer membrane are transient and areusually dispensed with once they have catalyzedtheir localization function. Thus, removal of targeting and localization elements at theamino-terminal end of the protein has been proposed to provide a vectorial 11lAAC- basis for their biogenesis in the bilayer. In its simplest form, DHFR the localization of proteins utilizes different determinants at A B C D E F G H the amino-terminal end of the protein which are dispensed FIG.6. Mitochondrial subfractionation of ATPI, full- with following their utilization. The ADP/ATP carrier protein of the mitochondrial inner length AAC, and 11 1AAC-DHFR fusion proteins. Mitochondria and reticulocyte lysate containing "'S-labeled proteins were incubated membrane is representative of a class of integral membrane in a buffer containing 0.6 M mannitol/HEPES/KOH (pH 7.4) (see proteins which are localized within mitochondria. First, there "Experimental Procedures") for 30 min at 30 "C. Mitochondria were are no apparent post-translational processing steps which reisolated (lanes A, supernatant; lanes B , pellet), treated with 250 pg/ participate in its localization pathway. Second, the extreme ) for 30 min at 0 'C,and again reisolated (lanes ml proteinase K (PA. C, supernatant; lanes D, pellet).Mitochondria were resuspended amino-terminal endof the ADP/ATP carrierdoes not exhibit under hypotonic conditions (0.1 M mannitol, 10 mM Tris (pH 8.0), 1 an aminoacid sequence with the potential for forming strucmM PMSF) for 10 min a t 0 "Cand reisolated (lanes E, supernatant; tures whichhavebeendocumented previously to mediate lanes F,pellet). The pellet was resuspended in 0.6 M sucrose, 3 mM protein import (Hurt et al., 1984; Allison and Schatz, 1986; MgCI,, 3 mM ATP, 10 mM Tris(pH 7.4) buffer, sonicated, and centrifuged to sediment membranes (lanes G, supernatant; lanes H, Vassarotti et al., 1987). Earlier work from this laboratory has pellet). Aliquots from each fraction were analyzed by gel electropho- shown that the first 115 residues of the AAC protein are resis and autoradiographed. IMS, intermembrane space. sufficient to deliver a hybrid gene product into mitochondria

ADPIATP Import Carrier Protein (Adrian et al., 1986). In this study, in uitro import analysis of AAC-DHFR hybrids revealed that the first 72 amino acid residues of the carrier alone were not sufficient for binding to mitochondria; however, a hybrid containing 111 residues of the carrier protein exhibited import which was as efficient as that for the full-length AAC protein. A striking featureof the sequence of the AAC protein between residues 72 and 111 is the predicted transmembrane a-helix located between residues 71 and 97 and a basic amphiphilic region between residues 98 and 111 (Saraste and Walker, 1982; Bogner et al., 1986; Adrian et al., 1986). Whether this region of the AAC protein alone is necessary for binding andtransportinto mitochondria or if it requires the interaction of sequences more amino-distal in the protein remains to be determined. Recently, the mitochondrial import of a Neurospora crussa ADP/ATP carrier protein lacking the amino-terminal 103 amino acids has been demonstrated (Pfanner et al., 1987). In this study, the authors demonstrate that the presence of an energized inner membrane is required to locate the carrier 104-313 protein to a space protected from 250 Fg/ml proteinase K. Under these assay conditions, this would locate the protein at stage 5 within the inner membrane (Pfanner and Neupert, 1987b). Several possibilities are possible to explain the apparent difference between our study and these observations. First, the ADP/ATPcarrier proteins aredocumented to be composed of three homologous domains of about 100 residues, each of which is apparently derived from a common ancestor (Saraste and Walker, 1982). Both studies are consistent with the possibility that each third of the AAC protein may contain sufficient information for binding to the mitochondrial surface and internalization.This would predict that each third of the translocator would contain sufficient information for binding and internalization at least to stage 3, where it would be protected from protease, yet clearly would be an intermediatein the import pathway (Pfannerand Neupert, 198%). In thisregard, each third of the AAC protein contains the hydrophobic domains aswell as putative amphiphilic regions to bind and initiateimport at or near the import site (Schwaiger et al., 1987). Clearly, a construct harboring two parts of this tripartite proteincan be fully imported (Pfanner et al., 1987). The AAC protein appears to entermitochondria via a class of high affinity saturable binding sites which also participate in the binding of porin to theouter mitochondrial membrane (Pfaller and Neupert,1987). Characterization of the 111AACDHFR protein indicated that it can only partially complete import and does not passbeyond stage 3 in the carrier protein import pathway. The demonstration of this intermediate is the first example of a stable intermediate in a mitochondrial import pathway which accumulates in an organelle which is completely competent for import. This intermediate, which was unable to pass beyond stage 3, occurred even though energized membranes capable of efficiently importing the asubunit precursor were present. The stage 3 intermediatewas characterized as aprotease-protected form of the AAC protein which was not inserted inthe membrane in a transmembrane form. The import block of the 111AAC-DHFR protein at stage 3 could be a function of either the lack of participation by sequences distal to the first 111 residues or interference by the soluble DHFR domain on the activity of the first 111 residues beyond stage 3. We have observed, however, that an AAC peptide consisting of just the first 115 residues of the protein is localized as an intermediate in the same manner as the 111AAC-DHFR protein in this study.* Therefore, we D. Caudle and M. Douglas, unpublished data.

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propose that theinability of the hybrid protein to insert into the bilayer beyond stage 3 was most likely due to the lack of additional sequences more carboxyl-terminal to first 111 residues, perhaps a second transmembrane-spanning sequence. It is noteworthy in this regard that the insertion of M13 procoat protein across the bilayer into a transmembraneform requires two hydrophobic membrane-spanning sequences working together. Analysis of the insertion of the M13 procoat into the E. coli inner membrane has shown that theintroduction of mutations (Kuhn et al., 1986a) or deletions (Kuhn et al., 1986b) to remove or break up the second transmembranespanning domain severely delays the kinetics of or block entirely the membrane localization of the protein. We suspect that thesame principles documented for insertion of the M13 procoat protein into the bilayer may define the movement of the AAC protein into the bilayer beyond stage 3. Studies to define both the role of additional transmembrane-spanning domains in stable membrane insertion and the component to which the stage 3 intermediate is complexed are currently in progress. Acknowledgments-We wish to thank Gwen Horton for technical assistance and Raquel Voss for her help in the preparation of this manuscript. REFERENCES Adrian, G., McCammon, M., Montgomery, D., and Douglas, M. (1986) Mol. Cell. Bwl. 6, 626-634 Allison, D., and Schatz, G. (1986) Proc. Natl. A c Q ~Sci. . U. S. A. 83, 9011-9015 Aquila, H., Link, T., and Klingenberg, M. (1985) EMBO J. 4, 23692376 Bogner, W., Aquila, H., and Klingenberg, M. (1986) Eur. J . Biochern. 161,611-620 Casadaban, M., and Cohen, S. (1980) J. Mol. Biol. 138, 179-207 Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135 Chen, W.-J., and Douglas, M. (1987) Cell 49,651-658 Daum, G., Bohni, P., and Schatz, G.(1982) J. Biol.Chem. 257, 13028-13033 Douglas, M., McCammon, M., and Vassarotti, A. (1986) Microbiol. Rev. 50,166-178 Fujiki, Y., Hubbard, A., Fowler, S., and Lazarow, P. (1982a) J. Cell Biol. 93, 97-102 Fujiki, Y., Fowler, S., Shio, H., Hubbard, A., and Lazarow, P. (198213) J. Cell Biol. 93, 103-110 Gasser, S., Daum, G., and Schatz, G. (1982) J. Bid. Chem. 2 5 7 , 13034-13041 Hartl, F., Schmidt, B., Wachter, E., Weiss, H., andNeupert, W. (1986) Cell 47,929-951 Hase, T., Muller, U., Reizman, H., and Schatz, G. (1984) EMBO J. 3,3157-3164 Hurt, E., Pesold-Hurt, B., and Schatz, G. (1984) EMBO J. 3, 31493156 Hurt, E., Pesolt-Hurt, B,. Suda, K., Oppliger, W., and Schatz, G. (1985) EMBO J. 4, 2061-2068 Klingenberg, M. (1981) Nature 290,449-454 Klingenberg, M. (1985) in The Enzymes of Biological Membranes (Martonosi, A., ed) Vol. 4, pp. 511-553, Plenum Press, New York Kuhn, A., Kreil, G., and Wickner, W. (1986a) EMBO J. 5 , 36813685 Kuhn, A., Wickner, W., and Kreil, G. (1986b) Nature 322,335-339 Laemmli, u. (1970) Nature 227,680-685 Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Molecular Cloning: A Labratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY OMaIley, K., Pratt, P., Robertson, J., Lilly, M., and Douglas, M. (1982) J.Biol. Chem. 257, 2097-2103 Pfaller, R., and Neupert, W. (1987) EMBO J. 6 , 2635-2642 Pfanner, N., and Neupert, W. (1987a) Curr. Top. Bioenerg. 15, 177219 Pfanner, N., and Neupert, W. (1987b) J . Biol. Chem. 262,7528-7536 Pfanner, N., Hoeben, P., Tropschug, M., and Neupert, W. (1987a) J. Biol. Chern. 2 6 2 , 14851-14854

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6790

ADPIATPImport Carrier Protein

Pfanner, N., Muller, H., Harmey, M.,andNeupert, W. (1987b)EMBO J. 6,3449-3454 Pfanner, N., Hartl, F., Guiard, B., and Neupert, W. (1988)Eur. J. Biochem., in press Roise, D., Horvath, S. J., Tomich, J. M., Richards, J. H., and Schatz, G. (1986)EMBO J. 5 , 1327-1334 266,9037-9043 Runswick, M., Powell, S.,Nyren, P., and Walker, J. (1987)EMBO J. 6,1367-1373 Sanger, F.,Coulson, A., Barrell, B., Smith, A., and Roe, B. (1980)J. Mol.711 143, 161-178 Saraste, M., and Walker, J. (1982)FEBS Lett. 144, 250-254 Schwaiger, M.,Herzog, V., and Neupert, W. (1987)J . Cell BioZ. 105, 235-246

Simonsen, C., and Levinson, A. (1983)Proc. Natl. Acad. Sci. U. S. A . Takeda, M., Chen, W.-J., Saltzgaber, J., and Douglas, M. (1986)J . Biol. Chem. 261,15126-15133 Todd, R. D., Buck, M. A., and Douglas, M. G. (1981)J. Biol. Chem. Van Loon, A., and Schatz, G. (1987)EMBO J. 6,2441-2448 Van Loon, A., Bran&, A., and Schatz, G . (1986)Cell 44,801-812 Vassarotti, A., Stroud, R., and Douglas, M. (1987)EMBO J. 6,705von Heijne, G. (1986)EMBO J. 5 , 1335-1342 Zwizinski, C., Schleyer, M., and Neupert, W. (1984)J. Biol. Chem. 259. 7850-7856