THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Societyfor Biochemistry and MolecularBiology, Inc.
Vol. 262, No. 32, Issue of November 15, pp. 15605-15609 1987 Printed in C.S.A.
The Roleof Protein Structure in the Mitochondrial ImportPathway UNFOLDING OF MITOCHONDRIALLY BOUND PRECURSORS IS REQUIRED FOR MEMBRANE TRANSLOCATION* (Fbceived for publication, May 26, 1987)
Wen-Ji Chen and MichaelG. Douglas$ From the Department of Biochemistv, Southwestern Graduate School of Biomedical Sciences,The University of Texas Health Science Center a t Dall&, Dallas, Texas 75235
the signal sequence of the growing polypeptide emerging from In order to examine the influence of protein structure on the post-translational importof a protein into mi- the ribosome promotes initiation of the assembly of the signal tochondria, the carboxyl-terminal129 residues of F1- recognition particle. This event initiates a protein secretion ATPase&subunitprecursor (511aa) havebeen re- scenario in which further translationof the protein is arrested placed with 61 residuesofyeastcoppermetallothiin the cytoplasm until the nascent protein-ribosome complex onein. Importofthe F1 B-copper metallothionein binds to thereceptor on theendoplasmic reticulum membrane (BCuMT) hybrid into mitochondriawas as efficient as (Walter and Blobel, 1984). However, in the case of nascent that of the FI @ precursor in the absence of copper. proteins destinedfor the mitochondria synthesis of precursors Addition of copper to mitochondrial import reactions, in the cytoplasm does not promote an apparent translational which had no significant effect on import of the F1 8subunit precursor, blocked import of theBCuMT pro- arrest. This is most dramatically demonstrated by the observation in yeast that the use of antibiotics (Reid et al., 1982; tein. Thiscopper-dependenttransportblockforthe BCuMT precursoroccurred after theprecursor was Nelson andSchatz, 1979) or conditional mutations which bound to mitochondria. Expression of the BCuMT pro- block mitoohondrial import (Yaffe and Schatz, 1984; Yaffeet tein in vivo revealed that BCuMT would bind copper al., 1985) caused the accumulation of mitochondrial precurand allow growth of a copper-sensitive yeast host on sors in the cell. These accumulated precursors can be subsean otherwise inhibitory level of the cationas long as it quently chased into their mature mitochondrial forms. Further, thepost-translational import of proteins into mitochonwas localized in the cytoplasm. These data indicate that the binding of copperBCuMT by renders it refrac- dria in vitro has been documented in many studies. tile for partial unfolding which is necessary for its Nascent and completed mitochondrial precursor proteins translocationintomitochondria.Theseobservations to the can form tertiaryandquaternarystructuresprior provide an alternative scheme for the selection of mu- initiation of theirproteintransport (Chen and Douglas, tants defective in mitochondrial import. 1987a). Therefore, additional factors or activities maybe required at the membrane surface of mitochondria for their import competency. Indeed recent studieshave demonstrated that the post-translational import of the Fl-ATPase’ @-subAnalysis of the protein signals which direct the localization unit precursors (Pfanner and Neupert, 1986; Eilers et al., of nascent proteinsto various intracellular compartments has 1987; Chen and Douglas, 1987b) requires ATP hydrolysis at revealed a clear distinction between those which catalyze the mitochondrial membrane surface in addition to a memmembrane insertion into the endoplasmic reticulum versus brane potential. The current model for this requirement is those which transport proteins into mitochondria (Wickner that ATP hydrolysis promotes the “reorganization” of the and Lodish, 1985). The signals defining import of proteins mitochondrial precursor to make it competent for import into mitochondria are distinguished by an extreme amino(Pfanner and Neupert, 1986; Eilers et al., 1987; Chen and terminal region which is hydrophilic, usually basic, and caDouglas, 198713). pable of forming secondary structure with an amphipathic Since a system may beassociated with mitochondria to deal character (Douglas et al., 1986; Roise et al., 1986; VonHeijne, with mitochondrial precursors which have formed structure 1986). Remarkably, the presence of a mitochondrial import due to their completion, it should be possible to demonstrate signal at the amino terminus of any soluble protein is suffithat structures which may be refractile to unfolding will in cient to catalyze the transfer of that protein into the mitochondrial matrix (Hurt et al., 1984; Honvich et al., 1985; Emr turn be blocked for import into the mitochondria. Earlier studies from this laboratory have demonstrated thatthe et al., 1986). In thecase of protein delivery to theendoplasmic reticulum, expression of a large hybrid precursor consisting of the Fl@subunit at the amino terminus and the Escherichia coli lacZ * This investigation was supported by National Institutes of Health at thecarboxyl terminus caused a block in the completion of Grants GM26713 and GM36537. The costs of publication of this protein translocation into mitochondria. This was presumably article were defrayed in part by the payment of page charges. This due to theformation of an active ATP2-lacZ tetramer on the article must therefore be hereby marked “advertisement” in accordmitochondrial surface which cannot be rendered import comance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of Grant 1-814 from The Robert A. Welch Foundation. petent (Douglas et al., 1984). To examine the effect of precursor protein structure on its Submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree. To whom correspondence should be addressed Dept. of Biochemistry, Southwestern Graduate School of Biomedical Sciences, University of Texas Health Science Center a t Dallas, 5323 Harry Hines Blvd., Dallas, T X 75235.
The abbreviations used are: F,-ATPase, the water-soluble portion of the yeast mitochondrial ATPase complex located on the matrix face of the mitochondrial inner membrane; kb, kilobase pairs.
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Analysis of ATP2-CUP1 Gene Fusions
import competency, a recombinant has been constructed between the yeast gene ATP2 encoding the F1 @-subunitprecursor and CUPl. The yeast CUPl encodes a copper metallothionein protein, a small cysteine-rich protein, 61 residues in length. This protein chelates copper and is proposed to participate in the detoxification of heavy metals in the cell (Winge et al., 1985; Monia et al., 1986). This protein has been purified from yeast (Winge et al., 1985); however, structural studies have not been completed. Secondary structure predictions (Butt et al., 1984) suggest that the dominant structure of the yeast protein in the absence of metals is P turn. The yeast protein is homologous with the metallothionein from rat liver whos? structure as the metal chelated form has been resolved at 2 A (Furey et al., 1986). This analysis has revealed that the protein forms a roughly-spherical structure with a diameter of approximately 15-20 A. In the present study the carboxyl-terminal 129 residues of the @-subunit precursor have been replaced by the coding region of CUP1 (61 amino acids). We have examined the behavior of this protein for mitochondrial protein import, both in vitro and in vivo. These studies show that in vitro protein translocation of the @-subunitcopper metallothionein precursor is the same as that for the @-subunitprecursor in the absence of copper ions but exhibits a greater sensitivity to copper for import both in vivo and in vitro. These observations therefore areconsistent with a model that some mitochondrial precursors can form a structure which is refractile to unfolding to prevent their import into mitochondria. EXPERIMENTALPROCEDURES
Strains and Media-The E. coli strain MC1066 (Casadaban and Cohen, 1980) used for plasmid transformation was grown inLB medium (Miller, 1972). Ampicillin-resistant transformants were selected in LB medium supplemented with 50 pg/ml of ampicillin (Miller, 1972). Yeast strain D273-10B MATa was used for mitochondrial preparations. The cells were grown in semisynthetic media as described in the accompanying paper (Chen and Douglas, 1987a). Yeast strain AB17 (Mat a, arg4-8, leu2-3, leu2-112, his7-2, trpl-289, ura3-52, ade5, cupl”) (Ecker et al., 1986) was used for in vivo analysis of differentATP2-CUP1 constructs.The cells were grownin YPD media (Sherman et ul.,1982), and the transformantswere selected in minimal media supplemented with all amino acids minus uracil (Sherman et al., 1982). Plates containing copper were prepared in the same media supplemented with indicated concentration of CuS04. Plasmid Constructions-Construction of plasmid containingATP2 and CUPl adjacent to the T7 promoter in pT7-1 is summarized in Fig. 1. A 0.3-kb RsaI fragment which contains 61 amino acids of CUPl coding region and 30-base pair 5‘-noncoding sequences was first isolated from YEp36 (a gift from Dr. R. Butow, University of TexasHealth Science Center at Dallas) (Butt et al., 1984). The fragment was then ligated into the HincII site in the polylinker of the plasmid pT7-1 to generate the plasmid, pT,-CUPl. In thesecond step, the 1.4-kb EcoRI-BarnHI fragment that covers the aminoterminal 382 amino acids and 310-base pair 5’-noncoding sequences of ATP2 was isolated from pT7-ATP2 andinserted into pT7-CUP1. This plasmid was designated pT?-PCuMT. The ATP2-CUP1 gene fusion in this plasmid codes for a 53-kDa fusion protein which contains 382 amino acids of F1-ATPase @-subunit at the amino terminus and 61 amino acids of yeast copper metallothionein at the carboxy terminus. These two domains are connected by a stretch of 13 amino acids derived from the polylinker sequences and the 5’noncoding sequences of CUPI. In a similar manner a fusion protein containing a presequence deletion in ATP2 which was unable to be imported into mitochondria was prepared. The 1.4-kb EcoRI-BarnHI fragment of pT7-flCuMT was replaced by an EcoRI-BarnHI fragment isolated from pT7-f14437 (Vassarotti et al., 1987). The obtained plasmid was named pT,PA4-37CuMT. In order to prepare plasmids for analysis of the different ATP2-CUP1 hybrids in uiuo, yeast E. coli shuttle vectors were prepared in the following manner: the SstI-Hind111fragments of pT7-
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FIG. 1. Construction of plasmid pT,
[email protected] transcription plasmid pT7-@CuMTwas constructed in two steps. First, a 0.3kb RsaI fragment containing CUPl from YEp36 (Butt et al., 1986) was ligated into the HincII digested transcription plasmid pT,-1 (Chen and Douglas, 1987). Next, the resulting plasmid pT7-CUP1 was digested with EcoRI and BarnHI. A 1.4-kb EcoRI-BamHI fragment containing 382 amino acids of the F, @-subunitplus 310 bp of 5”flanking DNA from pT,-ATP2 (Chen and Douglas, 1987b) was ligated into pTT-CUP1 with polylinker sequences and CUPl5’ DNA at the fusion joint. PCuMT and pT7-PA4-37CuMT were moved in place of the ATP2 SstI-Hind111 fragment in pj3A4-37. These yielded the yeast E. coli 2pm shuttle plasmids pBCuMT and pPA4-37CuMT. The gene fusions were moved as EcoRI-HindIII fragment into the polylinker site of pcSEY58 (Emr et al., 1986) to yield the CEN4 ARSl plasmids harboring the respective gene fusions (Fig. 4). Miscellaneous-Yeast strain AB17 was transformed by lithium acetate (LiAc) method (Ito et al., 1983). Cells were spread on yeast nitrogen base dextrose medium which maintained selection for uracil (Douglas et al., 1984). All otherExperimental Procedures are as described in the accompanying paper (Chen and Douglas, 1987a). RESULTS
To examine the effect of protein structure on the translocation of a mitochondrial precursor into the organelle, we constructed a chimeric protein by fusing the amino-terminal 382 amino acids of an imported mitochondria1 protein, F1 psubunit, to the 61 amino acids of copper metallothionein protein (see “Experimental Procedures,” Fig. 1).This ATPZCUPl construct yields a hybrid precursor protein with an apparent molecular weight of 53,000 when transcribed and translated in vitro, which should retain mitochondrial import determinants at its amino terminusand copper chelation activity at its carboxy terminus. The effect of copper on the import of the @-coppermetallothionein (PCuMT) protein into mitochondria was examined in an in uitro reaction by incubating precursor with isolated mitochondria and the indicated concentrations of copper. As
Analysis of ATP2-CUP1 Gene Fusions
15607
shown in Fig. 2, import of the @CuMT precursor was inhibited 4 O -30° a t a concentration of copper which had no discernible effect on the import of the F, @-subunit precursor. Quantitationof cu2+ these import reactions indicated that half-maximal inhibition P.K. of the @CuMT precursor occurred a t approximately 35 PM copper, whereas half-maximal inhibition of the wild type F1 {hubunit precursor occurred at 90-100 p ~ At . acopper -P T concentration of 75 PM, import of the @-copper metallothim onein precursorwas completely blocked. These results clearly revealed that replacing the carboxyl-terminal 129 residues of the8-subunitprecursorwiththe coding region of CUP1 specifically reduced the import efficiency of this protein in the presence of copper ion. PCuMT 1 The effect of copper on the importof the @CuMT precursor could occur by one of twomechanisms. First, copper may a globular copper interact with theprecursortogenerate FIG. 3. Partial unfolding at the mitochondrial surface is metallothionein chelate which is unable to partially unfold required to complete protein translocation across the memfor import. Alternatively, copper may complex to the @CuMT brane. Labeled F, p and pCuMT precursors were incubated with precursor toyield a structure which renders the mitochondrial energized mitochondria a t 4 "C in the absence of copper for 20 min. Following this, 75 p~ Cu2+was added and the reactions were incuimport signal inaccessible for binding at the mitochondrial bated for an additional 10 min. Valinomycin (0.4 p ~ was ) added to import site. In order to distinguish between these possibilities, each reaction priortoshiftingthetemperatureto 30 "C for an a mitochondrial import reaction with either theF1 @-subunit additional 20 min. Proteinase K protection analysis was performed precursor or the @CuMT precursor was initiated a t low tem- as described in the legend to Fig. 2. perature (4 "C) in theabsence of added copper. Previous studies have demonstratedthatmitochondrialimportcan initiate correctly a t low temperaturebutnotcomplete (Schleyer and Neupert, 1985). When the temperature of the import reactionwasraised to 26-30 "C, mitochondrially bound precursorswill complete their import.As shown in Fig. 3, when the bound precursors are warmed to 30 "C, import and maturationof the proteins into the mitochondrial matrix is observed. However, addition of copper to the bound precursors at 4 "C prior to the warm-up specifically inhibited the import of @CuMT precursor. We conclude from this result that theeffect of copper on the @CuMT precursor to prevent Srl I + Hmd I11 srt I Hind I11 itstranslocation is presumably due to the formation of a globular structure on the @CuMT precursor which is refractile to unfolding. This result further implies that protein unfolding must occur on the surface of mitochondria in order to complete its translocation across thebilayer. @-Copper Metallothionein Retains Its Copper Binding ActiuEcoRI L4 ity in Viuo-In order to confirm that theeffect of copper on import of the @-copper metallothionein protein in vitro was indeed due to the chelation of copper to the: protein, we examined the influence of copper on cells able to express the @-coppermetallothionein constructionin uiuo. Yeast plasmids were constructed containing the PCuMTsequence as well as a (EuMT-coding region lacking amitochondrial importsignal
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PCuMT FIG. 2. Addition of Cu2+to an in vitro mitochondrial import reaction blocks transport of the j3CuMT but not the F,j3 precursor. [""SIMethionine labeled F, p precursor (8)and OCuMT were incubated with energized mitochondria as described under "Experimental Procedures."Copper attheconcentrationsindicated was included at the beginning of each incubation. Following incubation a t 30 "C for 30 min, each import reaction was divided in half and further incubated with (+) or without (-) proteinase K; P.K., proteinase K; p., precursor protein; m, mature protein.
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FIG. 4. Construction of shuttle vectors for expression of j3CuMT and j3A4-37CuMT proteins in vivo. The plasmid ppA437 encodes the F, 8-subunit protein with a deletion between codons 4 and 37 (Vassarotti et al., 1987). A gene fusionbetween ATP2 harboring the64-37 deletion and CUP2 was constructed as described for ATP2 in Fig. 1 to yield pT7-pA4-37 CuMT. For each CUP2 gene fusion an SstI-Hind111 fragment was moved in place of the ATP2 SstI-Hind111 fragment of ATP2 in pPA4-37. These yielded the yeast E. coli 2-pM shuttle plasmids ppCuMT and ppA4-37 CuMT. The gene fusions were moved as EcoRI-Hind111 fragments into the polylinker site of pcSEY58 (Emr et al., 1986) to yield the CEN4 ARSl plasmids harboring therespective gene fusions.
(flA4-37CuMT, see Vassarotti, et al., 1987). The two gene fusions were ligated into yeast 2 pm and centromereplasmids which could be maintained in yeast strain AB17 by Ura+
15608
Analysis of ATP2-CUP1 Gene Fusions
selection (Fig. 4). These plasmids were transformed into the Cu2+ the PCuMT hybrid is blocked from further entry into yeast copper-sensitive strain AB17 which exhibited a sensi- mitochondria and should accumulate in the cytoplasm and tivityto growth inthepresence of copper of 10-20 p ~ . raise the tolerance of the host to Cu2+.This behavior of the Analysis of the growth properties of AB17 harboringthe PCuMT hybrid i n vivo is currently being utilized with the different constructs in the presence of increasing concentra- appropriate CUPl chromosomaldeletion host to select for tions of copper revealed a sensitivitytothemetal which mutations in yeast which are much reduced in their import reflected both the level of hybrid gene product synthesized efficiency. These mutants areselected for their ability to grow on a fermentable carbon source in the presence of elevated and its localization in the cell (Fig. 5). Cuz+ concentrations. Mutants selected in this manner are Growth of AB17 transformants containing the pPCuMT was inhibited on 20 p~ to the same extent as control trans- currently being characterized for conditional defects in mitoformants of AB17 containing thepcSEY58. Only transform- chondrial import. ants which expressed the PCuMT harboring thepresequence DISCUSSION deletion (PA4-37CuMT) on a multicopy plasmid were able to grow in the presence of 20 pM copper. Expression of the same Metallothionein isa small cysteine-rich metal-binding proconstruct harboring a functional presequence, pPCuMT, was tein which is ubiquitous in nature. Structural, spectroscopic, sensitive to 20 ptM Cu2+. We suggest that this sensitivity to and biochemical studies on a variety of mammalian metalloCuz+ is due to thelocalization of the hybrid PCuMT protein thioneins have demonstrated that the chelationof transition in the mitochondrial matrix away from the cytoplasm where metals by this protein causes its folding into a double domain it could sequester the toxic cation. Expression of the pA4- globular structure(Furey et al., 1986). X-raycrystaldata 37CuMT protein ona centromere plasmid did not apparently indicate that each of the !wo domains is roughly spherical yield sufficient hybrid to protect the hostfrom 20 p~ Cu'+. with a diameter of 15-20 A (Furey et al., 1986). In addition, Growth of yeast on a nonfermentable carbon sourcecaused the amino terminus of the highly homologous metallothionein a 3-fold increase in the level of FIB-subunit protein in mito- from rat liver is exposed as the metal chelate (Furey et al., chondria. This increase was due in part to an elevated level 1986), thus making this protein an attractive candidate for of transcription (Szekely and Montgomery,1984). When the present analysis. The present study was initiated to take AB17 contained thePA4-37 CuMT constructwe observe that advantage of tight metal binding propertyof the protein and slightly higherconcentrations of copper are required to inhibit the well-defined globular structure which it formsupon metal growth on a nonfermentable carbon source (not shown). As chelationto probe the effect of proteinstructureonthe noted previously for the behavior of these constructs in the transport of a protein into mitochondria. host on a fermentable carbon source (Fig. 5), only the preseSeveral studies indicate that at least partial unfolding of a quence deletion (A4-37) which maintained a cytoplasmicform protein is required for its transport intomitochondria. When of the hybrid allowed growth in the presence of 50 p~ Cu2+. translocation of the F1-ATPase @-subunitprecursor was The behavior of the PCuMT hybrid proteinsin vivo supports "trapped" during i n vitro import by incubation at 4 "C, a the observation that they bind Cu2+.The presenceof PCuMT translocational intermediate was documented which spanned hybrid in the cytoplasm allowed yeast to grow on otherwise both the inner and outer mitochondrial membranes (Schleyer inhibitory level of Cu2+. and Neupert, 1985). The amino-terminal end of the protein It is noteworthy that the presence of a functional mitowas processed by the metallo-proteaselocated withinthe chondrial presequence on the amino terminus which caused mitochondrialmatrix while thecarboxyl-terminalendrelocalization of the protein to the mitochondrial matrix does mained accessible to proteinase K from the outside. This not protect the hostfrom increased concentrationsof copper. translocationalintermediate, which could complete import This observation is consistent with previous studies indicating following a shift of the reaction to 30 "C, must have existed that during steady-state growth in yeast there are no detect- in a partially unfolded state during transport to exhibit siable cytoplasmic pools of mitochondrial precursors. Thus, the multaneous accessibility to the probes intwo compartments hybrid gene product PCuMT is being sequestered from the separated by two membranes. cytoplasm as it is being synthesized. This observation suggested that partial unfolding of mitoThe present studies are consistent with the model that chondrialprecursors occurred duringtheirpost-translocacompleted forms of the hybrid PCuMT proteins if present in tional entry into mitochondria. In a complementary study, the cytoplasm will complex with Cuz+.When complexed with formation of a structure in a completed protein prior to or after initiation of mitochondrial import blocked its translocation through themembrane. A hybrid protein containinga cytochrome oxidase subunit 4, COX4, presequence fused to mousedihydrofolatereductase could not be imported into mitochondria when the dihydrofolatereductase moiety is stabilized by the binding of inhibitor methotrexate (Eilers and Schatz, 1986). This result was interpreted as binding of the inhibitor prevented partial unfolding of the COX4-dihydrofolate reductase protein for import. In the present study, addition of Cu2+blocked import of the FIP-subunit precursor when its carboxyl-terminal 129 residues were replacedwith 61 residues of the CUPl gene product FIG. 5. Localization of PCuMT in the cytoplasm of yeast encoding the yeast copper metallothionein. Concentrations of of copper. Yeast the cation which inhibited the PCuMT hybrid had no effect renders it resistant to high concentrations strain AB17 transformed with the indicated plasmids were streaked on the post-translational import of the wild type P-subunit. ontoyeast nitrogen base dextrose plates.Twenty PM CuS04 was included in the plus copper plates. AB17 transformed with thefollow- Based on the demonstrated abilityof the PCuMT protein to of the ing plasmids were grown 3 days at 28 "C.A, pcSEY58; B, ppA4-37; bind Cu'+ i n vivo, we propose thattheinhibition C, pcBA4-37 CUMT;D,ppA4-37 CUMT,E , PcBCUMT;F, PDCUMT. mitochondrial import by the cation is due to the chelation
Analysis of ATP2-CUP1 Gene Fusions
15609
Casadaban, M., and Cohen, S. (1980) J. Mol. Bwl. 1 3 8 , 179-207 and formation of a tertiary structure which is resistant to Chamberlain,J. P. (1979) Anal. Biochem. 8 , 132-135 partial unfolding thatis required for transport. Chen, W.-J., and Douglas, M. G.(1987a) J. Biol. Chem. 2 6 2 , 15598Recent analysis of the in vitro import of the F,P-subunit 15604 precursor has demonstrated that ATPhydrolysis is required Chen, W.-J., and Douglas, M. G. (1987b) Cell 49,651-658 for the completionof protein translocation into mitochondria Douglas, M. G., McCammon, M. T., and Vassarotti, A. (1986) Microbwl. Rev. SO, 166-178 (Pfanner and Neupert, 1986; Eilers et al., 1987; Chen and Douglas, 1987). Data from these studies are consistent with a Ecker, D. J., Butt, T. R., Sternberg, E. J., Neeper, M. P., Debouck, model which evolvesthe utilization energy from phosphodies- C., Gorman, J. A., Crooke, S. T.(1986) J. Bwl. Chem. 2 6 1 , 1689516900 ter bond cleavage for the generation of a mitochondrially Eilers, M., and Schatz, G. (1986) Nature 322, 228-232 bound precursor that is competent for translocation. In the Eilers, M., Oppliger, W., and Schatz, G. (1987) EMBO J. 6 , 1073present context binding of Cu2+ to metallothionein is proposed1077 Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L., Takeda, M., and t o generate a structure which cannot be sufficiently reorgaDouglas, M. G. (1986) J. Cell Biol. 1 0 2 , 523-533 nized for a productive translocation event. Furey, W. F., Robbins, A. H., Clancy, L. L., Winge, D. R., Wang, B. Behavior of the PCuMT protein invivo, in addition to C., and Stout, C. D. (1986) Science 2 3 1 , 704-710 documenting the copper binding activity of the constructs, Gasser, S. M., Daum, G., and Schatz, G. (1982) J. B i d . Chem. 2 5 7 , also suggested the use of the ATP2-CUP1 gene in further 13034-13041 genetic analysis of the mitochondrial import pathway. The Horwich, A., Kalousek,F.,Mellman, I., and Rosenberg, L. (1985) EMBO J. 4,1129-1135 presence of a functional import signal in the ATP2-CUP1 Hurt, E. C., Hurt, B. P., and Schatz, G. (1984) EMBO J. 3 , 3149gene product does not alter in any significant manner the 3156 sensitivity of its cup" host to Cu2+ when expressed in uiuo. H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacterid. Similar analysisof the same construct but lacking a functional Ito,153,163-168 importsignalrendersthe cup" host resistant to a lethal Laemmli, U. K. (1970) Nature 2 2 7 , 680-685 concentration of copper. These datasuggest that thelocaliza- Maniatis,T., Fritsch, E.F., andSambrook, J. (1982) Molecular Cloning, p. 107, Cold Spring Harbor Laboratory,Cold Spring Hartion of the ATP2-CUP1 hybrid protein, in the case of the bor, NY PA4-37CuMT, places the metallothionein in the cytoplasm t o reduce the effective toxic concentrationof copper. If, how- Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory,Cold Spring Harbor,NY ever, the hybrid was localized in the mitochondrial matrix Monia, B. P., Butt, T. R., Ecker, D. J., Mirabelli, D. K., and Crooke, due to the presence of a functional import signal, then a high S. T. (1986) J. Biol. Chern. 2 6 1 , 10957-10959 sensitivity to cation wasnoted. This observation provides Nelson, N., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U. S. A . 7 6 , 4365-4369 further support for the contention that the synthesis and import of mitochondrial precursors are tightly coupled in uiuo, Pfanner, N., and Neupert, W. (1986) FEBS Lett. 209,152-156 Reid, G., and Schatz, G. (1982) J. Biol. Chem. 2 5 7 , 13062-13067 namely, that no free cytoplasmic precursor exists complex to D., Horrath, S. J., Tomich, J. M., Richards, J. H., and Schatz, copper, block import,andgenerate a Cu2+-resistanthost. Roise, G. (1986) EMBO J. 5, 1327-1334 These observations, therefore,suggest that host mutants se- Schleyer, M., and Neupert, W. (1985) Cell 43,339-350 lected on a fermentable carbon source which mislocalize the Sherman, F., Fink, G . R., and Hicks, J. B. (1982) Methods and Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory, PCuMT hybrid t o generate mutants resistant to Cu2+ may potentially define components of the import machinery. Stud- Cold Spring Harbor,NY ies are currently in progress to characterize such mutants. Szekely, E., and Montgomery, D. L. (1984) Mol. Cell. Biol. 4 , 939946
gratefully acknowledge Laura Vallier for her technical assistance and Jerry Lynn Allen for stimulating discussions during the course of this work. We would also like to thank Raquel Voss for her assistance in the preparation of this manuscript. Acknowledgments-We
REFERENCES Butt, T. R., Sternberg, E. J., Gorman, J. A., Clark, P., Hammer, D., Rosenberg, M., and Crooke, S. T.(1984) Proc. Natl. Acad. Sci. U. S. A . 31,3332-3336
Vassarotti, A., Chen, W-J., Smagula, C., and Douglas, M.G. (1987)
J.Biol. Chem. 262,411-418 Von Heijne (1986) EMBO J. 5,1335-1342 Walter, P., Gilmore,R., and Blobel, G. (1984) Cell 3 8 , 5-8 Wickner, W. T., and Lodish, H. F. (1985) Science 230,400-407 Winge, D. R., Nielson, K. B., Gray, W. R., and Hammer,D. H. (1985) J. Biol. Chem. 2 6 0 , 14464-14470
Yaffe, M. P., and Schatz, G. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1,4819-4823
Yaffe, M. P., Ohta, S., and Schatz, G. (1985) EMBO J. 4,2069-2074
