The Mitochondrial FIATPase a-Subunit Is Necessary for Efficient Import of Mitochondrial Precursors*

The mitochondrial import and assembly of the FIATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA’ and hsp70SSC’ and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the FIATPase a-subunit (ATPlp) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the FIATPase a-subunit (AATP1) versus mitochondria that lack the other major catalytic subunit, the FIATPase &subunit (AATPP). Yeast mutants lacking the a-subunit but not the &subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the Fl/3 subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1j3-subunit precursor accumulates as a translocation intermediate in absence of the Fla-sub- unit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the 8- subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the Fla-subunit likely functions as an “assembly partner” to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import

The Mitochondrial FIATPase a-Subunit Is Necessary for Efficient Import of Mitochondrial Precursors* (Received for publication, November 12, 1991) Huabing Yuan$ and Michael G. Douglas  The mitochondrial import and assembly of the FIATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA' and hsp70SSC' and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the FIATPase a-subunit (ATPlp) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the FIATPase asubunit (AATP1) versus mitochondria that lack the other major catalytic subunit, the FIATPase &subunit (AATPP). Yeast mutants lacking the a-subunit but not the &subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the Fl/3 subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1j3-subunit precursor accumulates as a translocation intermediate in absence of the Fla-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the 8subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the Fla-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.
Transport of mitochondrial precursors into mitochondria requires the sequential participation of proteins at the site where the inner and outer membranes are in close contact (reviewed in Attardi and Schatz 1988;Hart1 and Neupert 1990;Pfanner and Neupert, 1990;Douglas et al., 1991). The function of these proteins in the translocation complex is to maintain the precursors in an extended form during transit through the membrane (Pfanner et al., 1990). Following association of precursors with the mitochondrial surface, several * This work was supported by National Institutes of Health Grant GM36537. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ components have been identified which participate in translocation across both membranes. Initially, an integral outer membrane protein, ISP42, provides at least part of the translocation pore within the outer membrane of the contact site (Vestweber et al., 1989;Baker et al., 1990;Kiebler et al., 1990). The precursor protein is then transported through unknown components of the inner membrane to the mitochondrial hsp70ssc' protein on the matrix face of the inner membrane. The hsp70ssc1 protein most likely acts along with other proteins to clear precursor from the contact site and ISP42 and allows for the efficient transport of additional precursors (Kang et al., 1990;Scherer et al., 1990). The failure of either the ISP42 or hsp7OSSC1 proteins to properly function in yeast mitochondria causes accumulation of mitochondrial precursors and cell death (Kang et al., 1990;Vestweber et al., 1989;reviewed in Baker and Schatz, 1991).
Other proteins like hsp60'IF4 in mitochondria participate in addition to hsp70SSC1 in the assembly of different precursors. The groEL-like protein hsp60'IF4 acts in assembly of macromolecular complexes within the organelle matrix during transport mediated by hsp70ssc' (Cheng et al., 1989). Furthermore, studies of nuclear pet mutants which appear to be specifically required for formation of the mitochondrial ATPase complex implicate their role as well in specific assembly events within the organelle matrix (Bowman et al., 1991;Ackerman and Tzagoloff, 1990). Similar properties have been ascribed to another yeast pet mutant, ABC1, which affects cytochrome b complex assembly in mitochondria (Bousquet et al., 1991).
Recent reports have proposed that the mitochondrial FIATPase' a-subunit is an hsp60-like protein (Luis et al., 1990) with assembly properties like a molecular chaperone (Avni et al., 1991). The participation of molecular chaperones in protein import in addition to the observation that assembly events within mitochondria might be mediated by the Flasubunit prompted us to examine the role of this subunit directly. The availability of yeast mutants which specifically lack different F'ATPase subunits allowed us to examine the role of this subunit in protein import.
Yeast mutants lacking the mitochondrial Fla-subunit grow at considerably slower rates than mutants harboring deletions of other mitochondrial respiratory complex and ATPase subunits (see "Results"). Thus, this subunit may influence growth of yeast in some manner other than as part of an ATP synthetase. In this study, we show that mutants missing the mitochondrial Fla-subunit exhibit a delay in the import of some mitochondrial precursors which is not observed in mitochondria missing the FIB-subunit. Data are provided which The abbreviations used are: F,ATPase, soluble portion of the ATPase complex bound to the mitochondrial inner membrane; GIP, general insertion protein; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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Assembly of the FIATPase a-Subunit
show that the loss of Fla-subunit most directly affects the efficiency of the FIP-subunit which in turn interferes with the import of other precursors.
DNA Techniques-Transformation of E. coli strain MC1066 and preparation of small scale plasmid DNA were as described (Maniatis et al., 1982). Restriction digests were as described by the commercial suppliers. Linearized plasmid DNA for in vitro transcription was extracted twice with ch1oroform:phenol (l:l), once with chloroform:isoamyl alcohol (24:l). DNA was then precipitated with 70% ethanol and isolated by centrifugation. The resultant pellet was rinsed with 70% ethanol, dried, and then resuspended in 10 mM Tris, pH 7.4, made 1 mM in EDTA at 1 pg/yl. I n Vitro Transcription and Translation-The genes coding for precursors to F,ATPase @-subunit, pT7ATP2 (Chen and Douglas, 1987a), the F,ATPase a-subunit, pT7ATP1 (Takeda et al., 1986), and the ADP/ATP carrier protein, pT7AAC1 (Smagula and Douglas, 1988) under control of the phage T7 polymerase promoter were as previously described. The plasmids, pTqATP2, pT7ATP1, and pT7AAC1 were, respectively, linearized with HindIII, BamHI, and EcoRI. Plasmids pT7COX5a and pT,COX4 were provided by Michael Cumsky (Irvine CA). 2-5 pg of linearized plasmid DNA was added to transcription reactions containing T7 polymerase (Promega Corp., Madison, WI) as described earlier (Chen and Douglas, 1987a). Products of transcription reactions were extracted twice with chloroform:phenol (l:l), once with ch1oroform:isoamyl alcohol (24:l). mRNA was then precipitated by addition of sodium acetate (300 mM) and ethanol (70%) and isolated by centrifugation. The resultant pellet was rinsed with 70% ethanol, dried, and then resuspended in 50 p1 of RNase free H, O (Maniatis et al., 1982). Purified mRNA transcripts (5 pl) were used immediately in translation reactions or were stored frozen at -80 "C. Biosynthesis of 35S-labeled precursor protein from mRNA transcripts was carried out in nuclease-treated reticulocyte lysate exactly as instructed by the supplier (Promega Corp.) except Trans3'S-label (1059 Ci/mmol, ICN Biomedicals) was substituted for [:%]methionine. Translation reactions were used immediately in import reactions or were stored frozen at -80 "C.
Transport-competent Semi-intact Yeast Cells-Yeast cells were grown to early log phase in semisynthetic medium containing 2% of galactose and 0.1% of glucose. Semi-intact yeast cells were prepared from different mutants with minor modification of published methods (Baker et al., 1988). Spheroplasts were harvested by centrifugation at 4 "C, washed, and resuspended at 300 AGUO units /ml in lysis buffer (400 mM sorbitol, 20 mM HEPES (pH 6.8), 150 mM KOAc, 2 mM MgOAc, 0.5 mM EGTA) at 4 "C. Aliquots (200 pl) of the spheroplast suspension were transferred into Eppendorf tubes and frozen in the vapors above liquid N2 for 45 min and stored at -80 "C.
I n Vitro Import of Mitochondrial Precursors-In vitro import of the "'S-labeled mitochondrial precursors was performed in 50-pl reactions containing import buffer (200 mM sorbitol, 10 mM HEPES-KOH, pH 7.4, 100 mM KOAc, 1 mM MgOAc, 1.0 mM ATP, 10 mM succinate, 1 mM dithiothreitol, 25 mM creatinine phosphate, 50 pg/ ml creatine phosphate kinase), 1-3 pl of 35S-labeled precursor protein (in translation mix), and 12.5 yl of semi-intact cells. The import reaction was started by incubating the mix at 25 "C for 15-20 min. Following import cells were washed with 10 volumes of cold lysis buffer prior to analysis on 8% sodium dodecyl sulfate-polyacrylamide gels.
I n Vivo Pulse Labeling-Cells were grown in semisynthetic medium, 2% of glucose to early log phase. The cells were harvested and resuspended at 2.5 AmO units/ml in low sulfate medium plus 0.2% of glucose followed by incubating at 18 "C for 30 min. The cells were then pulsed for 3 min by addition of T r a n~~~S -l a b e l to 100 pCi per ml, and chased for 0, 0.3, 1, 3, and 5 min by adding cycloheximide to 40 pg/ml and 10 mM amino acids. Cell extracts were made from each sample by vortexing cells with glass beads. Extracts were processed for immunoprecipitation as previously described (Gasser et al., 1982). Antiserum to hsp6OMIF4 protein was kindly provided by A. Honvich (Yale University Medical School).
Miscellaneous-Cells were harvested at early log phase, and mitochondria were isolated as described (Gasser et al., 1982). Ten pg of mitochondrial protein from each strain was resuspended in 100 pl of mitochondrial import buffer. Each sample was divided in two parts: one-half was kept on ice and the other half was treated with proteinase K at 200 pg/ml for 30 min on ice. Western transfers to nitrocellulose were as previously described (Takeda et al., 1986).

RESULTS
In Vitro Import of Mitochondrial Precursors in the Absence of the Fla-Subunit-Participation of molecular chaperones in protein import and the chaperone-like properties proposed for the FIATPase a-subunit prompted us to directly examine the extent to which this subunit may participate in the assembly or transport of different precursors (Kang et al., 1990;Hart1 and Neupert, 1990;Glick et al., 1991). Assembly of the FIATPase within mitochondria is catalyzed by hsp6OMIF4 (Chang et al., 1989). In the present studies, washed semi-intact yeast cell preparations lacking either FIATPase subunit were used for import rather than isolated mitochondria. All measurable parameters of mitochondrial import were the same as in isolated mitochondria,' however, semi-intact yeast cell preparations from the deletion mutants were more stable and exhibited higher import efficiencies following storage in liquid nitrogen. We first examined in vitro import into mitochondria lacking either the Fla-(AATP1) or F& (AATPB) subunits (top panel, Fig. la). Import of the FIa-or F,@-subunit precursors was about the same into either wild type mitochondria or mitochondria lacking the p-subunit (compare lanes C and E ) . In contrast, mitochondria lacking the F,a-subunit (AATPl) failed to efficiently import the FIBsubunit precursor (Fig. la, lane G). It is noteworthy that much of the Fla and FIP precursor associated with mitochondria retained their presequence. This result indicated that loss of the Fla-subunit either substantially retarded translocation of the FIP precursor amino terminus into the matrix space or that the precursor amino terminus was associated with mitochondria such that it was not accessible to the matrix protease. Little mature FIP precursor (12% in this experiment) was noted in AATPl mitochondria (Fig. la, lane G, lower).
The import of presequence-containing precursors including the FIATPase precursors requires the presence of a membrane potential across the mitochondrial inner membrane. The inability of the AATPl mitochondria to efficiently import the FIP-subunit could not be ascribed entirely to a reduction or loss of the membrane potential. First, import of the Flasubunit and its processing in AATP1 mitochondria was little changed from wild type (Fig. la, lane G, upper). Import of the Fla-subunit into AATPl mitochondria was essentially the same as that of mitochondria prepared from the wild type H. Yuan and M. Douglas, unpublished observations. Nuclear pet mutants lacking the F,a-subunit (AATPI), FI&subunit (AATPZ), or the isogeneic wild type strain were grown in YP galactose media, and semipermeabilized cells were prepared according to Baker et ai., 1988. Import reactions contained 150 pg of whole cell protein in a total volume of 50 pl. h, import reactions programmed with the F&suhunit precursor a t zero time as descrihed in a were terminated at the times indicated. The relative amount of imported protein in each case was quantitated by laser scanning densitometry and plotted. wtATPIAATP2 was set at 100% (Ito et al., 1983).

Assembly of the F,ATPase a-Subunit
strain ( 8 8 f 10%; n = 5). Second, mitochondria from the AATP2 strain, which lacked the other subunit of the FIATPase complex exhibited little or no reduction in in vitro import of either Fla-, FIB-, or cytochrome oxidase subunit precursors (see Fig. 2). Third, as described below (see Fig. 4), mitochondria which lacked both ATPase subunits imported proteins nearly as well as wild type mitochondria.
The relative efficiencies for import of the FIB precursor into mitochondria lacking either the Flaor F&subunits was further determined by examining the kinetics of import (Fig.   lb). Import of FIB precursor into mitochondria lacking the 8subunit was almost complete in 10-12 min. Under the same conditions, mitochondria lacking the F1a-subunit exhibited a much slower rate of import. After a lag, the FIB precursor could be imported a t a reduced but constant rate over the time course of the experiment (Fig. 16). Eventually, the slow import into AATP1 mitochondria could reach a level which was about 50% that of maximal import into AATP2 mitochondria.
The import of other mitochondrial precursors were examined to determine if the import block imposed by the absence of the F,a-subunit was restricted to the ATPase precursor subunits. We observed that two other presequence containing mitochondrial precursors for the cytochrome oxidase complex, COX4 and COX5a, were inhibited to approximately the same extent as FIB (Fig. 2). The cytochrome oxidase complex, like the ATPase complex, constitutes a major inner membrane enzyme in mitochondria which is assembled from imported precursors.
In Vivo Transport of Mitochondrial Precursors in the Absence of the Fla-Subunit-In vitro studies indicated that the in vivo import rate of mitochondrial precursors should be delayed in the AATP1 mutant. In order to confirm this, we examined the in viuo import kinetics of two mitochondrial precursors in cells lacking the Fla-subunit. To directly compare the kinetics of import between AATP1 and ATP1 strains, we examined the import of the hsp60""' and FIB precursors. In a separate study, import of hsp6OMIF' in AATP2 cells was indistinguishable from wild type (not shown). As shown in Fig. 3, the processing of both FIB and hsp60""' precursors was decreased in the AATP1 strain. Pulse labeling revealed that the rate of hsp6O"IF' precursor import in AATP1 cells was decreased by a t least &fold. We observe that the tnh for h~p60"'~ import into wild type mitochondria (<0.3 min) increased to 2 2 min in AATP1 cells. The kinetic effect on import was greater than the reduction in the rate of import observed in a MAS70 deletion host.' The MAS70 protein has been shown to accelerate the delivery of proteins into mitochondria (Hines et al., 1990).
Yeast mutants deleted for the Flcr-subunit exhibited a much slower rate of growth than other nuclear petites when grown on a fermentable carbon source (Table I)   AATPl host was attributable solely to the absence of the Finsubunit since transformation of the AATPl host with a plasmid containing the ATPl gene (pATP1) restored normal growth (Table I). In addition, point mutations in A T P l which prevent catalytic function of the assembled F,ATPase, atpl-1 , do not exhibit a slower growth rate (Takeda et al., 1986, Table I). This mutant has been shown to contain an assembled F,ATPase particle with less than 5% the specific activity of wild type (Tzagoloff et al., 1975).
Fla-Subunit and Import-The import delay observed in vitro and in oioo in mitochondria lacking the Fln-subunit could be explained by two general mechanisms. First, the Finsubunit may be participating as a chaperone for the import of different mitochondrial precursors. Although, this subunit does exhibit characteristics of a molecular chaperone we consider this possibility less likely. Second, the Fln-subunit may be indirectly participating by providing an "assembly partner" to allow more efficient import of the slowly imported FIB precursor. This possibility is more likely since import of the Fi@-subunit is much slower than that for other precursors (Reid and Schatz, 1982) and could effect general protein import if its import were delayed. In order to distinguish between these alternatives a yeast strain was constructed which removed both the Flaand Fij3-subunits (see "Experimental Procedures"). We then asked if mitochondria lacking both subunits were more efficient for import than those lacking the Fln-subunit. First, we observed that growth of the double deletion strain was restored to that of the typical nuclear petite strain such as one lacking the Fij3-subunit alone AATPP (Table I). Second, loss of the FI@-subunit (AATPlAATP2) improved the initial import rates in mitochondria already lacking the Fla-subunit almost 10 fold (Fig.  4, compare AATPlAATP2 to AATPlwtATP2 in the lower panel). The initial rate of import of Fij3-subunit precursor into mitochondria from the double deletion mutant was indistinguishable from either wild type or AATP2 (not shown) mitochondria. Third, import of other mitochondrial precursors was similarly restored in mitochondria lacking both subunits (not shown). Thus, in the absence of the Fln-subunit. the presence of the FI@-subunit affects protein import. Precursors Associated with AATP1 Mitochondria-The block in assembly imposed by the absence of the Fin-subunit could lead to the formation of in oitro translocation intermediates which are not normally visualized unless special conditions are used. There are examples where a defect in the assembly or processing of a mitochondrial precursor in the mitochondrial matrix leads to the formation of translocation intermediates. One intermediate which accumulates under conditions of matrix ATP depletion is a full length precursor associated with mitochondria in the intermembrane space which remains protected from protease (Hwang et al., 1991). In Fig. 5, protease digestion conditions were adjusted to determine the presence of translocation intermediates during import into AATP1 mitochondria. Under conditions in which the import rate of the FIj3-subunit precursor into mutant mitochondria is reduced (lower p a n e l , closed circles), we ob-  5. The presence of translocation intermediates during import into AATP1 mitochondria. Import reactions programmed with F,@-subunit precursor were performed as described in Fig. 1 for the times indicated.
Following import the semi-intact cells were washed and divided in half. One half was treated with 100 mg/ml proteinase K a t 0 "C for 25 min. This level of proteinase does not digest precursors protected only by the outer membrane. FIG. 6. FIb-subunit precursor is isolated bound to mitochondria from the AATPl mutant but not from the wild type strain. Mitochondrial samples (20 p g ) prepared from the LATl'1 mutant and wild type strains following growth on semisynthetic 2% galactose, 0.5% dextrose were treated with proteinase K (200 pg/ml).
Following protease treatment, digested (+) and control (-) samples were resolved on an SDS gel and probed with FI/j-suhunit and AA Carrier antiserum. In this experiment 43% of the F&suhunit ASSOciated with AATPl mitochondria was in the precursor form. serve a small but constant amount of the full length precursor is associated with AATPl mitochondria which is not digested by proteinase K (see gel panel, AATPlwtATP2, 3-15 min time points). This protected precursor likely results from rate limiting import imposed by the absence of the Fla-subunit.
The import delays observed in AATP1 mitochondria suggested that we might be able to detect precursor associated with isolated mitochondria. Normally, the small amount of precursor associated with mitochondria is rapidly turned over and is not observed in isolated preparations (Fig. 6, lunes A   and B). However, when mitochondria were prepared from the yeast lacking the Fla-subunit, we were able to detect associated FI/3 precursor. The level of precursor subunit associated with mitochondria represented a substantial fraction (40-48%; n = 5) of the total F,P-subunit present in mitochondria (Fig. 6). The precursor associated with AATPI mitochondria remained accessible to protease (lunes C oersuq D). The accessibility of this precursor to proteinase K could result from its association with mitochondria across the contact site but not far enough to be cleaved by the matrix-processing enzyme. Alternatively, the accessibility to proteinase K might reflect the association of precursor with receptor elements on the outer membrane such that it was accessible to protease.

DISCUSSION
Assembly of the F,ATPase in mitochondria occurs from five different kinds of subunits which are synthesized as precursors and imported to the matrix space of mitochondria. The localization of these subunits involves the participation of hsp7Oq"'" and h~pG0"'"~' to complete, respectively, translocation and assembly (Kang et al., 1990;Scherer et al., 1990;Cheng et ul., 1989). These molecular chaperones appear to act on the majority of macromolecular assemblies in mitochondria. In addition, other proteins have been proposed to transiently participate in the assembly of specific complexes. The products of ATP11 and ATP12 for example are proposed to participate in the assembly of the ATPase complex but are not part of the reconstitutively active enzyme (Ackerman and Tzagoloff, 1990).
Assembly of mitochondrial complexes occurs after the import and processing of their constituent subunits (Manning-Krieg et al., 1991). Mutants of h~p70"~"' block translocation and processing and this is proposed to be an early event at the matrix face of the inner membrane (Kang et al., 1990;Scherer et al., 1990). Mutations in hsp60""', on the other hand, located within the matrix can also block import into mitochondria, however, these data were not addressed in the original paper (Cheng et ai., 1989). Likewise, nuclear mutations which block the removal of presequences in the mitochondrial matrix eventually cause precursors to accumulate at the mitochondrial outer membrane (Yaffe and Schatz, 1986). These observations support a pathway for import of mitochondrial precursors into the matrix in which hsp70"'"'' functions in conjunction with the matrix presequence processing enzyme and the hsp6OV"" folding machinery for efficient precursor entry (Ostermann et al., 1989;Neupert. et ul., 1990;Manning-Krieg et ai., 1991).
In the absence of the Flcr-subunit, we observe kinetic delays in protein import as well as precursor associated with the surface of isolated mitochondria. These observations are consistent with either a direct action of the Fln-subunit on the import of different precursors or an indirect effect which results in interference for entry of other subunits. We observe that a deletion of the F1/3-subunit alleviates the import block and slow growth imposed by the absence of the Fln-suhunit. Since we find the F&subunit precursor associated with mitochondria lacking the cr-subunit, we believe that the Flnsubunit is required at some point for efficient import of the F,P-subunit.
Where in the import pathway does the absence of the Flnsubunit effect general import and what does this say about the coupling of import and assembly? The data provided here are most consistent with a mechanism in which the Flnsubunit acts at the point of assembly of the F,ATPase. In the absence of assembly, the FIB precursor accumulates on mitochondria such that it is accessible to proteases on the outside. The association of F,/3 precursor with isolated mitochondria lacking the F,n-suhunit supports the intervention of the Flnsubunit a t a point after the membrane potential-dependent