Oct 5, 1990 - crosslinked to a chimeric precursor protein composed of the following three ..... incubation for 30 min on ice with 0.5 mg/ml proteinase K; 3,.
The EMBO Journal vol.9 no.13 pp.4315-4322, 1990
A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein Philipp E.Scherer, Ute C.Krieg, Sam T.Hwang1, Dietmar Vestweber2 and Gottfried Schatz Biocenter, University of Basel, CH-4056 Basel, Switzerland, 'Brigham and Women's Hospital, Brookline, MA 02146, USA and 2MPI for Immunebiology, D-7800 Freiburg, Germany Communicated by G.Schatz
We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP - Sepharose chromatography and partially sequenced. Fourteen of its 15 N-terminal amino acids were identical to residues 24-38 of the protein encoded by the nuclear gene SSCI, which had been proposed to encode a dnaK-like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix-targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non-ionic detergent Triton X-100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space. Key words: matrix protein/mitochondrion/stress protein/ yeast
Introduction Import of cytoplasmically made precursor proteins into mitochondria is mediated by proteins in the cytosol, the mitochondrial membranes, and the matrix (reviews: Verner and Schatz, 1988; Pfanner and Neupert, 1990). The cytosolic components include 70 kd heat shock proteins that appear to accelerate import by preventing premature tight folding, or aggregation, of precursor proteins (Deshaies et al., 1988; Murakami et al., 1988). Similarly, a 60 kd heat shock protein related to bacterial groEL protein mediates refolding of precursors in the matrix space (McMullin and Hallberg, 1987, 1988; Cheng et al., 1989; Ostermann et al., 1989). Less is known about the identity and the functions of the membrane-associated components of the import machinery. We have recently identified a 42 kd protein of the yeast mitochondrial outer membrane that appears to be part of the
© Oxford University Press
import machinery of the outer membrane (Vestweber et al., 1989). This protein was specifically and efficiently photocrosslinked to a chimeric precursor protein composed of the following three parts: (i) a N-terminal matrix-targeting sequence; (ii) mouse dihydrofolate reductase (DHFR); (iii) bovine pancreatic trypsin inhibitor (BPTI) crosslinked to the C-terminal cysteine of DHFR. The crosslinker not only connected DHFR and BPTI, but also contained a photoactivatable diazirin group. When this chimeric protein was added to isolated energized yeast mitochondria, it became stuck across both mitochondrial membranes. The DHFR moiety was at least partly translocated into the matrix, the matrix-targeting sequence was cleaved off by the matrixlocalized processing protease, while the BPTI domain remained outside the mitochondria because its three intramolecular disulfide bridges prevented translocation (Vestweber and Schatz, 1988a; Vestweber et al., 1989). As mitochondria accumulating this precursor became importincompetent, the precursor appeared to jam the mitochondrial protein import machinery. In order to identify additional components of the mitochondrial protein translocation machinery, we have now crosslinked a stuck precursor to adjacent partner proteins not via a precursor-borne diazirin group, but via externally added, cleavable bifunctional crosslinkers. We detected crosslinking of the stuck precursor to a 70 kd protein which proved to be located in the matrix. The protein was purified, partially sequenced and found to be identical to the product of the previously identified nuclear SSCI gene which encodes a mitochondrial 70 kd heat shock protein (hsp70; Craig et al., 1987, 1989). This protein is synthesized with a typical N-terminal matrix-targeting sequence, is imported and cleaved by isolated mitochondria, and is similar in sequence to the other 70 kd stress proteins of the yeast cytosol (Craig et al., 1989). The protein is essential for viability, but its function has remained open. As our data indicate that mitochondrial hsp70 (mhsp7O) binds firmly to a translocation intermediate stuck in the import machinery, it may participate in one of the earliest steps leading to the folding of newly translocated precursors in the matrix space. The fact that mhsp7O is essential for viability is in line with our earlier suggestion (Yaffe and Schatz, 1984a,b) that components mediating key steps in mitochondrial protein import are necessary for the life of the yeast cell even in the absence of respiration. Such proteins include the two subunits of the matrix-localized processing protease (Yaffe and Schatz, 1984a,b; Yaffe et al., 1985; Yang et al., 1988), mitochondrial hsp60 (Cheng et al., 1989), and ISP42 (Vestweber et al., 1989; K.P.Baker, A.Schaniel, D.Vestweber and G.Schatz, in press). The present study suggests that mhsp7O is a fifth essential component of the mitochondrial protein import machinery.
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Results
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The translocation-arrested precursor can be crosslinked to at least one mitochondrial protein When the purified, radiolabeled COXIV-DHFR fusion protein (Figure 1, lane 1; 'pc') was coupled via its C-terminal cysteine to BPTI, most of it was converted to the chimeric COXIV-DHFR-BPTI adduct (Figure 1, lane 2; 'pc-BPTI'). During incubation with energized yeast mitochondria, both the chimeric precursor and the residual underivatized fusion protein were imported and most of the imported molecules were processed to their respective 'mature' forms ('mf-BPTI', 'mf'; Figure 1, lane 3; see also Vestweber and Schatz, 1988a,b; Vestweber et al., 1989). As shown earlier, the chimeric adduct becomes stuck across the mitochondrial membranes, jamming the protein import sites (Vestweber et al., 1989). When mitochondria that had accumulated the stuck chimeric precursor were solubilized with Triton X-100 and the extracted proteins crosslinked with the cleavable, homobifunctional crosslinker DSP, subsequent analysis by SDS-PAGE and fluorography revealed two crosslinked species of apparent molecular weights 125 000 and 105 000 (Figure 1, lane 4, arrowheads; labeled A and B on the right). These products were not seen if the chimeric precursor had been presented to deenergized mitochondria (Figure 1, lane 5). This suggests that formation of the crosslinks required partly translocated precursor stuck in the import site. With the apolar crosslinker DSP, the efficiency of crosslinking was essentially the same in intact mitochondria and in Triton extracts (Figure 2, lanes 1 and 2). In contrast, a polar DSP derivative, DTSSP, only generated the
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Fig. 2. Formation of the crosslinked products in intact mitochondria requires a membrane-permeable crosslinking reagent. The apolar crosslinker DSP generates the crosslinked bands (labeled A and B on the right) both in intact mitochondria (lane 1) and in mitochondria solubilized by Triton X-100 (lane 2). In contrast, the membraneimpermeable crosslinker DTSSP generates the crosslinked bands only in solubilized (lane 4) but not in intact (lane 3) mitochondria. Import of the chimeric precursor into energized mitochondria and analysis of the reaction products was as described in Figure 1. The samples were then immunoprecipitated with antibodies raised against the BPTI moiety of the chimeric precursor (see Materials and methods).
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Fig. 1. The translocation-arrested precursor can be crosslinked to at least one mitochondrial protein. Lanes: 1, 35S-labeled COXIV-DHFR fusion protein (10% standard); 2, chimeric precursor (COXIV-DHFR coupled to BPTI; 10% standard); 3, chimeric precursor imported into mitochondria; 4, same as 3, except that a Triton X-100 extract of the mitochondria was subjected to crosslinking with DSP; 5, chimeric precursor was incubated with mitochondria deenergized by 1 AM valinomycin, and a Triton X-100 extract of the mitochondria was crosslinked with DSP. Mr, molecular mass standards (sizes in kd). Samples were analyzed by SDS- 10% PAGE followed by fluorography. I, import; val, valinomycin; pc and mf, uncleaved and cleaved ('mature') form of the COXIV-DHFR fusion protein; pcBPTI and mf-BPTI, uncleaved and cleaved form of the chimeric COXIV-DHFR-BPTI adduct (the underivatized COXIV-DHFR variant DV12 undergoes processing in two steps; see also Vestweber et al., 1989). The arrowheads in lane 4 mark the crosslinked products A and B. The calculated molecular masses of the various radioactive species depicted in the figure are as follows: pc-BPTI, 31 kd; mfBPTI, 27 kd; pc, 25 kd; mf, 21 kd. 4316
crosslinks in Triton extracts (Figure 2, compare lanes 3 and 4). This observation suggests that in intact organelles, the crosslink occurs in an environment shielded from the aqueous suspension medium. The results presented below support this conclusion. The crosslinks occur in a complex containing the chimeric precursor and ISP42 When mitochondria containing the stuck intermediate were crosslinked with DSP and then solubilized by hot SDS in
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Fig. 3. The crosslinked products contain DHFR, BPTI and ISP42. The two crosslinked products (labeled A and B on the right) were generated as described in Figure 1, lane 4 and subjected to immunoprecipitation with antisera against BPTI, mouse DHFR, or ISP42. Where indicated ('SDS+'), the Triton extract was heated for 5 min at 95°C after addition of SDS to 2 % to disrupt non-covalent protein - protein interactions. Samples were analyzed by SDS PAGE using a 7-12% gradient gel. -
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order to disrupt any non-covalent protein -protein interactions, the radiolabeled crosslinked products were recognized by antisera against BPTI and mouse DHFR, but not by those against ISP42 (Figure 3, lanes 2, 4 and 5). However, upon solubilization under non-denaturing conditions with Triton X-100, immunoprecipitation was also seen with antibody against ISP42 (Figure 3, lane 6). The crosslinks are thus generated in a non-covalent complex containing the chimeric precursor and ISP42. DSP can apparently not crosslink ISP42 to the chimeric precursor under our experimental conditions. In any event, the observed covalently linked products (105 and 125 kd) are much larger than a possible covalent complex between the chimeric precursor (31 kd) and ISP42 (42 kd), and they should therefore contain at least one other A A
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Fig. 4. The translocation-arrested precursor is crosslinked to a 70 kd protein. Mitochondria (150 Ag) were allowed to import the chimeric precursor. They were then radioiodinated with Na['25I] by the chloramine-T method (Greenwood et al., 1963), solubilized by Triton X-100, and subjected to crosslinking with DSP. Precursor-containing material was enriched by immunoprecipitation with affinity purified anti-BPTI IgGs and visualized by SDS-8% PAGE and fluorography (1st dimension gel; note that non-crosslinked precursor has electrophoresed off this gel). The two crosslinked species (bands A and B) were excised, the crosslinks were cleaved by incubating the gel pieces for 30 min at 95°C in SDS-sample buffer containing 125 mM DTT, and the cleavage products generated from band A or B were analyzed on a second gel (SDS - 12% PAGE; second dimension). The cleavage products are marked as 'pc-BPTI', 'mf-BPTI' (see legend to Figure 1), '70 kd', and by an asterisk. 0 v .4
protein of higher molecular mass, presumably a mitochondrial protein. Both crosslinked bands contain a 70 kd mitochondrial protein In order to identify the mitochondrial protein(s) which had become covalently linked to the stuck translocation intermediate, we took advantage of the fact that the crosslinks could be cleaved by reducing agents such as DTT. Mitochondria were allowed to accumulate the translocation intermediate and then were radioiodinated to high specific activity by the chloramine-T method. The crosslinked products were then enriched by immunoprecipitation with anti-BPTI antibody followed by SDS -PAGE under nonreducing conditions (Figure 4, first dimension). Individual radioactive bands were excised from the gel, treated with DTT, and analysed by SDS -PAGE and fluorography in a second SDS -PAGE step (Figure 4, second dimension). Both crosslinked bands contained the cleaved and uncleaved form of the chimeric precursor; in addition, both the 125 kd (band A) and 105 kd (band B) crosslinked material consistently yielded a 70 kd cleavage product, whereas band B in some experiments yielded a 70 kd as well as a lower molecular mass cleavage product (Figure 4, asterisk). The crosslinked 70 kd protein is a matrix-located stress protein As the translocation intermediate had previously been shown to be in contact with the outer membrane protein ISP42 (Vestweber et al., 1989), we initially suspected that the 70 kd protein, too, was a component of the outer membrane. Accordingly, we separated outer membrane proteins by
SDS-PAGE, excised the polypeptides with relative molecular masses between 65 and 80 kd, and raised antibodies against the mixture of these proteins. These antibodies could indeed immunoprecipitate both crosslinked products (not shown). We then resolved outer membrane proteins by anion exchange FPLC in the presence of the nonionic detergent octyl-polyoxyethylene, coupled the individual protein peaks to CNBr-activated Sepharose beads, and used these beads to affinity purify the corresponding antibodies from the original polyvalent antiserum. Each affinity purified
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Fig. 5. Immune electron microscopy localizes the crosslinked 70 kd protein to inner mitochondrial compartments. Isolated yeast mitochondria were exposed to hypertonic conditions to shrink the matrix compartment, fixed and embedded, and thin sections were decorated with IgGs affinity purified according to their ability to immunoprecipitate the crosslinked bands. Bound IgGs were visualized by protein A-gold as detailed by Pon et al. (1989).
4317
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P.E.Scherer et al.
antibody fraction was tested for its ability to precipitate the crosslinked products. Although this procedure was laborious, it yielded an antibody preparation that both immunoprecipitated the crosslinked products and reacted with only a single 70 kd spot on a two-dimensional gel of outer membranes (not shown). To our surprise, immune electron microscopy indicated that the 70 kd protein recognized by this purified antibody was located inside the inner membrane (Figure 5). This suggested the possibility that the protein was a matrixlocated 70 kd stress protein; a member of this protein family (termed BiP) was recently shown to mediate protein translocation across the endoplasmic reticulum from the trans-side of that membrane (Vogel et al., 1990), and yeast contains a mitochondrial 70 kd stress protein (encoded by the nuclear SSCI gene) which is essential for viability (Craig et al., 1987). Accordingly, we purified a 70 kd ATP binding protein from the yeast mitochondrial matrix fraction to homogeneity (Figure 6A), and raised a monospecific antiserum against the protein. Both antibody preparations-the monospecific antibody that had been affinity purified based on its ability to immunoprecipitate the crosslinked products, and the antibody raised against the purified 70 kd matrix protein-decorated the same spot on a two-dimensional gel of outer membrane proteins (not shown). To prove that the 70 kd protein recognized by the two antibodies was indeed mhsp70, we subjected the purified protein to Edman degradation. Fourteen of its N-terminal 15 amino acids were identical to those predicted for positions 24-38 of the SSCI gene product (Figure 6B). The difference (E instead of the predicted Q in position 1 of the mature protein) might simply reflect posttranslational modification accompanying removal of the presequence as a Q - E change is also found in position 1 of mature cytochrome oxidase subunit IV from yeast (Maarse et al., 1984). As noted before, the protein's sequence is also A. Mr
similar to that of dnaK, an Escherichia coli stress protein (Tilly et al., 1983). If the 70 kd mitochondrial protein crosslinked to the stuck precursor is mhsp70, an antiserum raised against purified mhsp70 should precipitate the two crosslinked products. Figure 8A shows that this is indeed the case. We conclude that the 70 kd mitochondrial protein is mhsp70. The stuck precursor forms a tight complex with mhsp7O even without crosslinking To test whether the mhsp7O -precursor complex was stable enough to be isolated by co-immunoprecipitation without A. 2
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Fig. 6. Mhsp7O. (A) Purification: starting from 90 g (wet weight) of yeast cells, 500 jig of mhsp70 were obtained (Materials and methods). Lanes: 1, mitochondria (150 /g protein); 2, matrix fraction (150 Mg
protein); 3, flow-through of Zn-chelate column (50 MAg); 4, ATP-agarose eluate (2 Mg); 5, flow-through of DE-52 column (4 Mg). Fractions were analyzed by SDS 12% PAGE and staining with Coomassie Blue. 'mhsp7O', mitochondrial hsp 70. (B) N-terminal amino acid sequence of mhsp70 determined directly in this work (top line) compared with the deduced amino acid sequences of the SSCI gene product (Craig et al., 1989; middle line) and the Ecoli dnaK protein (Bardwell and Craig, 1984). -
4318
Fig. 7. Mhsp7O is located in the matrix. (A) Subcellular fractionation. Yeast cells were fractionated into a low-speed pellet ('nuclear pellet'), mitochondria, a high-speed pellet ('microsomes') and a high-speed supernatant (cytosol). Each fraction was analysed by SDS- 12% PAGE and immunoblotting with IgGs which had been affinity purified from crude serum (Materials and methods) according to their ability to immunoprecipitate the two crosslinked products. Identical results were obtained with antiserum against purified mhsp7O. The blots were also decorated with antisera against hexokinase (cytosol marker) and porin (mitochondrial marker). (B) Submitochondrial fractionation. Yeast mitochondria (4 mg) were suspended in 4 ml of 0.6 M sorbitol, 20 mM HEPES-KOH pH 7.4 and divided into four aliquots. The aliquots were treated as follows: 1, incubation for 30 min on ice; 2, incubation for 30 min on ice with 0.5 mg/ml proteinase K; 3, conversion to mitoplasts followed by incubation for 30 min on ice with 0.5 mg/mI proteinase K; 4, conversion to mitoplasts and incubation for 30 min on ice with 1 % octyl-polyoxyethylene plus 0.5 mg/ml proteinase K. All samples were then adjusted to 1 mM phenylmethyl sulfonylfluoride (PMSF) and aliquots were analysed by SDS- 12% PAGE followed by immunoblotting with antisera against the following proteins: MAS70 (outer membrane marker), cytochrome b2 (intermembrane space marker), hsp 60 (matrix marker) and mhsp 70. The radiolabelled bands were quantified by scanning of the fluorograms and their intensities expressed as % of the corresponding signal in the untreated mitochondrial sample (lanes 1).
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crosslinking, mitochondria containing the stuck chimeric precursor were either crosslinked with DSP or left untreated. They were then solubilized with Triton X-100 in the presence or absence of hot SDS, the extracts were subjected to immunoprecipitation with antisera against BPTI or ISP42, A.
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Fig. 8. The translocation-arrested chimeric precursor forms a stable complex with mhsp7O. (A) Crosslinking studies. The two crosslinked species were generated as described in Figure 1, lane 4. Proteins in the crosslinked Triton extracts were then denatured by adding SDS to 2% and heating for 5 min at 95°C, the denatured mixtures were diluted 20-fold with TNET and subjected to immunoprecipitation with antisera against either BPTI (lane 1) or mhsp7O (lane 2). (B) Coimmunoprecipitation of mhsp7O without crosslinking. The chimeric precursor (pc) was incubated with energized (Val-) or deenergized (Val+) mitochondria as described in Materials and methods except that each import reaction contained 0.8 mg mitochondria and 5 Ag of unlabeled chimeric precursor (variant KSAD) in a final volume of 0.5 ml. Crosslinking with DSP (DSP+) was performed in intact mitochondria. Samples were then solubilized either with 2 ml of Triton X-100-containing (TNET) buffer at room temperature (-SDS) or heated for 5 min at 95°C in 0.1 ml TNET containing 2% SDS followed by dilution with 1.9 ml TNET (+SDS). Samples were subjected to immunoprecipitation with the indicated antisera and the immunoprecipitates were analyzed by SDS - 10% PAGE under reducing conditions followed by immunoblotting with 125I-labeled IgGs against mhsp70. Lane 10 contains 10 ng of purified mhsp7O as a standard. aBPTI, camhsp7O, cxISP42, antisera raised against the corresponding proteins.
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and all immunoprecipitates were subsequently analysed for the presence of mhsp70 by SDS -PAGE and immunoblotting with radioiodinated IgGs against mhsp70. The complex between the stuck chimeric precursor and mhsp7O could be isolated even without crosslinking (compare lanes 1 and 4 of Figure 8B), provided the mitochondria were solubilized under non-denaturing conditions (compare lanes 4 and 5). In fact, DSP appeared to stabilize < 10% of all precursor-mhsp7O complexes by covalently linking its components (compare lanes 1 and 2). Thus, crosslinking did not significantly increase co-immunoprecipitation of mhsp7O by either anti-BPTI or anti-ISP42 antibodies (note the similar intensities of the immune signals in lanes 1, 4 and 6). Figure 8 confirmed that co-immunoprecipitation of mhsp7O by ISP42 antibodies only occurred if mitochondria had accumulated the stuck precursor (compare lanes 6 and 8). As expected, SDS abolished this co-immunoprecipitation (lane 7). Taken together, these data indicate that the stuck precursor is directly or indirectly associated with ISP42 in the outer membrane, while already bound to the matrix component mhsp7O. Association of mhsp7O with the stuck precursor does not require removal of the precursor's presequence In the experiments reported so far, most of the stuck precursor molecules were proteolytically processed by the matrix-located processing protease (e.g. Figure 1, lane 4). In order to test whether removal of the precursor's Nterminal matrix-targeting signal was a prerequisite for association with mhsp70, the chimeric precursor was presented to energized mitochondria whose matrix protease had been inhibited by 1,10-phenanthroline and EDTA (Figure 9, lane 3). As a control, Figure 9 also shows an import experiment in the absence of both chelators (lanes 1 and 2). The mitochondria were then treated with DSP, solubilized in SDS, and subjected to immunoprecipitation with antibodies against mhsp70. The immunoprecipitates were then analysed by SDS -PAGE under reducing conditions. After import into control mitochondria, most of the chimeric protein crosslinked to mhsp7O had been processed
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p/E Fig. 9. Precursor and mature form of the translocation-arrested protein crosslinked to mhsp70. The two crosslinked products were generated with DSP in intact mitochondria as described for lane 4, Figure 1 (lanes 1 and 4), except that some incubations contained valinomycin (lane 2) or 10 mM EDTA and 1 mM 1,10-phenanthroline (lane 3). Samples were immunoprecipitated under denaturing conditions with amhsp7O antiserum, and the immunoprecipitates were analysed by SDS- 12% PAGE in the presence of the reducing agent DTT. Lane 6: 1 % of the imported chimeric precursor as a standard. Val, valinomycin; p/E, 1, 10-phenanthroline/EDTA. are
Fig. 10. The transmembranous complex identified in this study. BPTI, bovine pancreatic trypsin inhibitor; ISP42, 42 kd outer membrane protein present in the import site; hsp70, mhsp7O; X, Y and Z, hypothetical additional proteins forming transport channels through the outer and inner mitochondrial membranes. 4319
P.E.Scherer et al.
(Figure 9, lane 1; note also that some of the processed form of the underivatized COXIV -DHFR had been crosslinked to mhsp7O). After import into mitochondria whose matrix protease had been inhibited, most of the radioactive chimeric protein recovered from the crosslinks was unprocessed (Figure 9, lane 3). Processing is thus not a prerequisite for association of an imported protein with mhsp7O. We conclude that this interaction most likely involves the 'mature' part of the precursor protein.
Discussion The experimental approach Photochemical crosslinking of membrane proteins to a partly translocated precursor has identified novel components of the protein translocation system associated with the endoplasmic reticulum (Wiedmann et al., 1987, 1989; Krieg et al., 1989) and mitochondria (Vestweber et al., 1989). Success of the method has hinged upon two factors: translocation of the precursor polypeptide was arrested at a specific stage, and the photo-crosslinker was present at specific sites on the precursor protein. While these conditions favour a high efficiency and specificity of crosslinking, they may limit the number of components that can be detected. Indeed, our previous photo-crosslinking experiments with mitochondria identified only ISP42 as a component located at the mitochondrial protein import site. In the present study we decided to retain the advantages offered by a precursor arrested at a specific stage of transmembrane movement, but to add reagents that could indiscriminately crosslink the precursor to adjacent components. The crosslinks generated by this method appeared to be specific for the following reasons. First, their appearance was dependent on a mitochondrial membrane potential, indicating that they originated from a bona fide translocation intermediate. Secondly, crosslinks were also obtained in essentially identical yields if the mitochondria containing the stuck precursors were solubilized by non-ionic detergent before crosslinking. Thus, the crosslinked products must have originated from a non-covalent, but relatively stable association of the stuck precursor with the mitochondrial protein import machinery. As the crosslinked bands were immunoprecipitated by ISP42 antibodies under nondenaturing conditions, but not in the presence of SDS, the complex apparently contained ISP42 which was not crosslinked to the precursor protein under the present
conditions. Identification of the 70 kd protein Initially we expected that the two crosslinked products represented crosslinks between the precursor and a 70 kd outer membrane protein. We could quickly rule out that this protein was MAS70 (Hines et al., 1990), as the crosslinked products were not recognized by anti-MAS70 IgGs and were still obtained with MAS70-deficient mitochondria (not shown). To our surprise, the protein proved to be a component of the matrix space. Its presence in isolated outer membranes must thus reflect a redistribution artefact. This artefact was avoided in experiments in which we probed the protein's submitochondrial location either by assessing its susceptibility to externally added protease in mitochondria and mitoplasts or by immune electron microscopy. A loca-
4320
tion in the matrix was further supported by the observation that, in intact mitochondria, the crosslinked bands were only generated by hydrophobic crosslinkers able to diffuse across the mitochondrial inner membrane; in Triton extracts, however, the crosslinks were generated in similar yields by membrane-permeant as well as membrane-impermeant crosslinkers. Definite characterization of the 70 kd protein was achieved by isolating it, sequencing its N-terminus, and identifying it as the product of the already known SSCI gene. In retrospect, our efforts to track down the protein's identity started in the wrong direction, but in the end they achieved their aim. The present work has thus identified a translocation complex extending from the mitochondrial surface across both mitochondrial membranes into the matrix space. A tentative model of this complex is shown in Figure 10. Function of mhsp70 in protein import Our experiments do not address the role of mhsp7O in mitochondrial protein import. However, two possibilities are suggested by the fact that the sequence of mhsp70 resembles that of the E. coli dnaK protein (Craig et al., 1989) which functions in the replication of DNA and as a chaperone protein (Liberek et al., 1988). While mhsp7O may well participate in the replication of mitochondrial DNA, this cannot be its only function: yeast cells are viable without mitochondrial DNA, yet mhsp7O is an essential protein. We therefore favor the view that the protein is part of the machinery that catalyzes refolding of polypeptides in the matrix space. These polypeptides might include products of the nuclear and the mitochondrial genetic system. As mentioned in the Introduction, there is already evidence to indicate that cytosolic hsp70 accelerates protein translocation across intracellular membranes and that a groEL-like 60 kd heat shock protein in the matrix catalyzes refolding of imported proteins. In both cases, additional components appear to be involved which are still unidentified. Mhsp7O may be such a component. This protein appears to be a ubiquitous mitochondrial protein as it has been detected not only in yeast, but also in protozoans (Engman et al., 1989; Searle et al., 1989) and mammals (Leustek et al., 1989; Mizzen et al., 1989; Amir-Shapira et al., 1990). As the protein is firmly bound to a precursor that still contains its cleavable matrix-targeting signal and that is still stuck across both mitochondrial membranes, interaction with mhsp70 may be one of the earliest, perhaps even the earliest, interaction of imported precursors with a matrix protein. Although the association may be triggered by the matrix-targeting sequence, this sequence is not required for the stability of the complex because the stuck chimeric precursor has already been processed to the 'mature' form. Association with mhsp7O is not limited to precursor molecules that are stuck in the import site as it is also observed with fully imported, underivatized COXIV-DHFR fusion protein (Figure 9, lane 1). Further work will be required to show whether mhsp70 transfers the bound polypeptide to some other component such as hsp60, whether it mediates a reaction independent of other chaperone-like catalysts, or whether it performs other, as yet unsuspected tasks. Our results implicating mhsp7O as a component of the mitochondrial protein import system may have opened a way for answering these
questions.
Yeast 70 kd mitochondrial stress protein
Materials and methods Import of the chimeric precursor into yeast mitochondria Most experiments employed the DV12 variant (Vestweber and Schatz, 1988b) of the COXIV-DHFR fusion protein (Eilers and Schatz, 1986); it was over-expressed in Ecoli cells labeled with [350S42 ] and purified as described (Vestweber and Schatz, 1988b). The radiolabeled fusion protein was chemically coupled via its unique C-terminal cysteine to BPTI that had been activated at one of its free amino groups by the heterobifunctional crosslinker m-maleimidobenzoyl-N-hydroxy succinimide ester (MBS). For experiments requiring larger amounts of precursor, we constructed, and isolated from overexpressing E. coli cells, a different version of the COXIV-DHFR fusion protein (termed KSAD). This protein, unlike DV12, still contains the original cysteine residue at position 7 of the DHFR moiety and is therefore more stably folded than DV 12. This permitted us to isolate milligram amounts of essentially homogeneous KSAD protein that could be selectively modified by MBS-activated BPTI at its C-terminal cysteine. The isolation protocol was that described for 'wild-type' COXIV-DHFR by Endo and Schatz, 1988. In general, mitochondria were isolated (Daum et al., 1982) from the Saccharomyces cerevisiae strain D273-IOB (ATCC 25657; MATce). For a control experiment with MAS70-deficient mitochondria (referred to in the Discussion), the S. cerevisiae strain HR-1 was used (Riezman et al., 1983). Unless stated otherwise, import assays contained 200 jg mitochondrial protein and 20 pmol (2-4 x 105 c.p.m.) of
radiolabeled chimeric precursor in a final volume of 400 /1 of 0.6 M sorbitol, 10 mM MgCI2, 25 mM KPi pH 7.4, 40 mM KCI, 0.5 mM EDTA, 25 mM Na-succinate, 25 mM Na-L-malate, 1 mg/ml of fatty aciddepleted bovine serum albumin, 0.5 mM ATP, 3.8 mM creatinine phosphate, 40 Ag/ml creatine kinase. Incubations were carried out for 15 min at 30°C, followed by reisolating the mitochondria by centrifugation in an Eppendorf Microfuge and washing them by resuspension in 400 /1 of buffer A (0.6 M sorbitol, 20 mM HEPES-KOH pH 7.4) and centrifugation.
Crosslinking After import, the washed mitochondria were suspended in 50 141 of buffer A or buffer B (0.5% Triton X-100, 0.5 M NaCl, 5 mM EDTA, 20 mM HEPES-KOH pH 7.4) and mixed with 2 u1 of a freshly prepared 5 mM solution of dithiobis(succinimidylpropionate) (DSP) or 3,3'-dithiobis (sulfonylsuccinimidylpropionate) (DTSSP) in dimethyl sulfoxide and incubated at 0°C. After 30 min, unreacted crosslinker was inactivated by adding L-lysine to 50 mM. The crosslinkers were purchased from Pierce
Chemical Co., USA. Immunoprecipitation For immunoprecipitation under non-denaturing conditions, samples
diluted with 1.1
were
of TNET (1% Triton X-100, 150 mM NaCI, 5 mM EDTA, 20 mM Tris-HCI pH 7.5). When samples were to be denatured before incubation with antibodies, they were heated for 5 min at 95°C in 50 ul 2 % SDS and then diluted to 1.1 ml with TNET. In most cases, 30 i1 of the desired antiserum or an appropriate amount of IgG solution were added and the samples were incubated at 4°C overnight. Immune complexes were adsorbed for 2 h at room temperature to Protein A -Sepharose beads (Pharmacia Co., Sweden; 50 ttl of a 1:1 (v/v) slurry of beads in TNET) and the beads were washed five times with a modified TNET solution whose concentration of Triton X-100 was only 0.05%. After a final wash with Triton-free TNET (i.e. NET), immune complexes were solubilized from the beads by heating them for 5 min at 95°C in 0.1 ml of SDS-PAGE sample buffer [3.6% (w/v) SDS, 15% (w/v) glycerol, 120 mM Tris-HCI pH 6.8]. When cleavage of the crosslinks was desired, the sample buffer also contained 125 mM dithiothreitol (DTT). ml
Purification of mhsp7O Mitochondria were prepared from a 10 1 culture of S. cerevisiae D273-1OB (Daum et al., 1982). One hundred g wet weight of cells yielded 728 mg (protein basis) of mitochondria. The matrix fraction was prepared from them (Bohni et al., 1983) and adjusted to 10 mM Tris-Pi pH 7.5, 1% Triton X-100 and 0.2 M NaCl (TTN buffer). This solution was passed through a Zn2+ chelate column (Pharmacia) that had been preloaded with 5 mg/mi ZnCI2 and then equilibrated with TTN buffer (Yang et al., 1988). The flow-through fraction was collected and passed through a Sephadex G-25 column (Pharmacia; bed volume 20 ml) which had been equilibrated with 20 mM HEPES-KOH pH 7.4, 10 mM K acetate, 2 mM Mg acetate, 2 mM DTT, (buffer C). Protein-containing fractions were pooled and loaded onto an ATP-agarose column (Sigma A-9264; bed volume 5 ml). The column was washed successively with 25 ml buffer C, 30 ml buffer C containing
1 M K acetate, 20 ml buffer C containing 0.1 M K acetate, and buffer C containing 0.1 M K acetate and 10 mM Mg2+ -ATP (buffer D). Proteins eluting in the presence of ATP were pooled and passed through a DEAE-cellulose column (Whatman DE-52) which had been equilibrated with buffer D. The flow-through contained essentially pure mhsp70 (Figure 6B).
Preparation of monospecific IgGs against outer membrane proteins Isolated mitochondrial outer membranes from yeast were solubilized in 20 mM Tris-HCI pH 8.0, 2% octyl-polyoxyethylene and loaded onto an anion exchange column (Superformance Fractogel-EMD-TMAE-650(S), Merck product Nr. 20286). The column was developed with a 0-0.3 M NaCl gradient in loading buffer and fractions were analyzed by SDS-PAGE and immunoblotting with antiserum raised against a mixture of 65-80 kd outer membrane proteins (see Results). The proteins of immune-reactive fractions were coupled to CNBr-activated Sepharose 4B (following the instruction of the manufacturer, Pharmacia) and the resulting affinity beads were used to bind specific IgGs. These were then eluted with 100 mM glycine-HCI pH 2.5 and neutralized with KP1 pH 8.0. They were tested for their ability to immunoprecipitate the crosslinked complexes and to decorate only a single spot on a 2-D gel of outer membrane proteins.
Miscellaneous Published methods were used for subcellular fractionation of yeast cells (Hase et al., 1984), submitochondrial fractionation (Daum et al., 1982), isolation of mitochondrial outer membranes (Riezman et al., 1983), conversion of mitochondria to mitoplasts (Daum et al., 1982), SDS-PAGE and fluorography (Hurt et al., 1984), immune blotting (Haid and Suissa, 1983), immune electron microscopy (Pon et al., 1989), production of antisera (Suissa and Reid, 1983), isolation of IgG by chromatography on Protein A-Sepharose (Hines et al., 1990), radioiodination of IgG by the chloramineT method (Greenwood et al., 1963) and assay of protein (BCA method; company brochure published by Pierce Chemical Co., USA).
Acknowledgements We are grateful to Dr Paul Jeno and Dr John Lambris for sequencing mhsp70, to Gaby Walker, Brigitte Marshallsay, Hildegard Brutsch, Wolfgang Oppliger and Kitaru Suda for excellent technical help, and to Marianne Jaggi, Liselotte Muller and Verena Grieder for the artwork. This study was supported by research grants from the Swiss National Science Foundation (3-26189.89), the US Public Health Service (2 ROI GM37803), the Human Frontier Science Program, and by fellowships from the European Molecular Biology Oranization (to U.C.K.) and the Samoff Endowment for Cardiovascular Science (to S.T.H.).
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Note added in proof Co-immunoprecipitation experiments have shown that 20-40% of the stuck precursor can be recovered in association with mhsp7O. Mhsp7O also binds to BPTI-free precursor that is completely imported into the matrix. In that case, however, the interaction is only transient. Morishima et al. [J. Biol. Chem., 265, 15189-15197 (1990)] have recently shown that mhsp70 is a functional component of a yeast mitochondrial endonuclease. Mhsp7O may thus have multiple functions.
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