©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Yeast Mitochondrial Protein Import Receptor Mas20p Binds Precursor Proteins through Electrostatic Interaction with the Positively Charged Presequence (*)

(Received for publication, October 21, 1994; and in revised form, December 8, 1994)

Volker Haucke (§) Trevor Lithgow (¶) Sabine Rospert Kerstin Hahne Gottfried Schatz (**)

From the Biozentrum, University of Basel, CH-4056 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein import into yeast mitochondria is mediated by the four outer membrane receptors Mas70p, Mas37p, Mas20p, and Mas22p. These receptors may function as two subcomplexes: a Mas37p/Mas70p heterodimer and an acidic complex consisting of Mas20p and Mas22p. To assess the relative contribution of these subcomplexes to precursor binding, we allowed different precursors to bind to the surface of deenergized mitochondria, then reenergized the mitochondria and measured the chase of the bound precursors into the organelles. Productive binding of several precursors with a positively charged amino-terminal matrix targeting sequence, such as SU9-DHFR, hsp60, and mitochondrial cpn10, was strongly inhibited by salt, by low concentrations of a mitochondrial presequence peptide, and by a deletion of Mas20p, but was independent of Mas37p/Mas70p. In contrast, productive binding of the ADP/ATP carrier was not inhibited by salt, the presequence peptide, or a deletion of Mas20p, but was strongly dependent on Mas37p/Mas70p. The precursors of alcohol dehydrogenase III and the Rieske iron-sulfur protein had binding properties between these two extremes. The productively bound precursor of cpn10 could be cross-linked to Mas20p. We conclude that Mas20p binds mitochondrial precursor proteins through electrostatic interactions with the positively charged presequence, whereas Mas37p/Mas70p may recognize some feature(s) of the mature part of precursor proteins.


INTRODUCTION

Import of proteins into mitochondria requires the specific recognition of cytoplasmically synthesized mitochondrial precursor proteins by receptor proteins in the mitochondrial outer membrane(1, 2) . Studies with Saccharomyces cerevisiae and Neurospora crassa have identified three such receptors: a 20-kDa protein termed Mas20p in yeast and MOM19 in N. crassa(3, 4) , a 70-kDa protein termed Mas70p in yeast and MOM72 in N. crassa(5, 6, 7) , and a 22-kDa protein termed Mas22p in yeast and MOM22 in N. crassa(8, 9) . A fourth receptor, a 37-kDa protein termed Mas37p, has so far been identified only in yeast. (^1)These four receptors exist as two hetero-oligomeric subcomplexes that may loosely interact with each other: a Mas37p/Mas70p heterodimer and an oligomer containing Mas20p and Mas22p(^2)(10) .

The relative contributions of these different receptor subunits to the overall import process have not been firmly established. Import experiments with isolated yeast mitochondria have suggested that different subsets of precursors differ in their dependence on each of the receptor subcomplexes. Mas37p/Mas70p promotes the import of the ADP/ATP carrier, cytochrome c(1), the F(1)-ATPase beta-subunit, and the mitochondrial isozyme III of the alcohol dehydrogenase, but not import of artificial precursors containing dihydrofolate reductase (DHFR) (^3)as a passenger protein. In contrast, Mas20p/Mas22p interact with most or all precursors containing mitochondrial presequences, including DHFR-containing fusion proteins(4, 7, 11, 12) . However, only Mas22p is essential for protein import and viability of yeast; Mas20p, Mas37p, and Mas70p can be deleted singly or in pairwise combination without blocking import as long as the level of Mas22p is maintained(9) .

It is still unknown how the import receptors specifically recognize precursor proteins destined for mitochondria. Experiments in which the presequences of authentic precursors were fused to DHFR have suggested that Mas20p/Mas22p, through their cytosolically exposed ``acid bristles,'' may bind the basic and amphiphilic mitochondrial presequences(9, 12) . Recognition by Mas37p/Mas70p appears to be more complex, involving a precursor's ``mature'' part(11) .

In this study we have attempted to determine which mitochondrial receptors specifically recognize NH(2)-terminal mitochondrial presequences. We found that the NH(2)-terminal basic and amphiphilic presequences are bound by the acid bristle complex containing Mas20p and Mas22p, that this binding is blocked by a chemically synthesized presequence peptide, and that it is predominantly mediated by a salt-sensitive electrostatic interaction. In contrast, binding of precursors to Mas37p/Mas70p is only partly inhibited by high salt or by a presequence peptide, confirming that this receptor subcomplex recognizes determinants in the mature part of precursor proteins.


EXPERIMENTAL PROCEDURES

Yeast Strains

All haploid S. cerevisiae strains were derived from the diploid strain YKB5 (MATalpha/a ura3/ura3 leu2/leu2 his4/his4 ADE2/ade2 LYS2/lys2). Experiments were performed with the following strains: the wild-type strain YTJB4 (MATa ura3 leu2 his4 lys2), the Deltamas37, Deltamas70 strain YSG3 (MATa ura3 leu2 ade2 mas70::LEU2 mas37::HIS4)(9) , and the Deltamas20 strain YTJB 17 (MATa ura3 leu2 his4 mas20::URA3)(13) .

Cell Growth and Isolation of Mitochondria

Cells were grown in 2% sodium lactate and 0.1% glucose, and mitochondria were isolated and purified as described(14) .

Binding and Chase Assays

To assay for productive binding and subsequent chase of precursors, 100 µg of mitochondrial protein was incubated for 5 min at 0 °C in 100 µl of buffer A (0.6 M sorbitol, 25 mM Hepes/KOH, pH 7.4, 12 mM KCl, 5 mM MgCl(2), 0.5 mM EDTA) containing 5 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Precursor binding and chase were done essentially according to (11) except that the chase was initiated by 10 mM DTT instead of 20 mg/ml bovine serum albumin. All samples were analyzed by SDS-PAGE and fluorography. To test for inhibition of productive binding by a presequence peptide, 100 µg of mitochondrial protein was incubated in 100 µl of buffer A containing 5 µM CCCP and up to 0.5 µM peptide dissolved in water.

Purification of His-tagged Yeast Mitochondrial Chaperonin 10 Expressed in Escherichia coli

A fragment containing the coding region of CPN10(15) was ligated into the plasmid pQE-60 (Diagen GmbH), and the resulting recombinant plasmid was used to transform the E. coli strain M15 (Diagen, GmbH). The M15 transformants were grown to an A of about 0.5, and expression of the His-tagged cpn10 was induced with 0.45 mM isopropyl-1-thio-beta-D-galactopyranoside for 2 h. The His-tagged cpn10 was purified under native conditions according to the manufacturer's instructions. The purity of the protein was greater than 90% as judged by SDS-PAGE and staining with Coomassie Blue. (^4)

Miscellaneous

Published methods were used for SDS-PAGE, standard DNA procedures, and transformation of E. coli, in vitro transcription/translation, import into isolated mitochondria(4, 20) , the generation of cross-links, affinity purification of antibodies, and immunoprecipitation(16) . The chemically synthesized rat liver mitochondrial hsp60 presequence peptide was a kind gift from Drs. N. Hoogenraad and P. B. Hoj (La Trobe University, Bundoora, Australia). The peptide was further purified by reverse-phase HPLC, and the purity and absolute amount of the eluted material were determined by quantitative amino acid analysis. Fluorograms were quantified using a computerized densitometer (Molecular Dynamics Co).


RESULTS

Productive Binding of Mitochondrial Precursors to the Surface of Yeast Mitochondria

Binding of precursors to mitochondrial outer membrane receptors does not require an electrochemical potential across the inner membrane, but import into or across the inner membrane absolutely depends on a potential(17) . We have used this differential energy requirement to study the productive binding of different precursors to mitochondria. We first incubated the radiolabeled precursors with yeast mitochondria that had been uncoupled with the protonophore CCCP. We then reenergized the mitochondria by inactivating CCCP and adding an energy source and chased the prebound precursor into the organelles. Finally, we assessed the effectiveness of the chase by determining the fraction of the prebound precursor that had become inaccessible to added protease. With precursors containing a cleavable presequence, we also measured proteolytic conversion to the mature form.

We chose the following precursors: a fusion protein containing the first 69 amino acids of the N. crassa F(1)F(0)-ATPase subunit 9 precursor fused to mouse DHFR (SU9-DHFR)(18) , mitochondrial hsp60, the mitochondrial isozyme of alcohol dehydrogenase III (pADHIII), the Rieske Fe/S protein, and the ADP/ATP carrier (AAC). These precursors were synthesized by coupled transcription/translation in a reticulocyte lysate and were used without further purification. Some experiments were also performed with chemically pure, radioiodinated yeast mitochondrial cpn10. This protein has an amphiphilic NH(2)-terminal matrix-targeting signal that is not cleaved upon import(15) .

When the radiolabeled precursors shown in Fig. 1A were incubated with deenergized mitochondria in a low-salt buffer, 10-30% of the added precursor molecules bound to the mitochondrial surface: the precursors remained accessible to added protease, and those with a cleavable presequence remained unprocessed. Upon a subsequent chase, between 25 and 50% of the prebound precursors could be chased into the mitochondria: they became protease-inaccessible, and any cleavable presequence was removed (Fig. 1A). Thus, between 25 and 50% of the bound precursor molecules were productively associated with the mitochondrial surface (11) .


Figure 1: Precursor binding to the mitochondrial surface. A, different precursors can be bound productively to the mitochondrial surface. The precursors of SU9-DHFR, hsp60, pADHIII and the ADP/ATP carrier (AAC) were translated in vitro and incubated for 5 min at 0 °C with isolated mitochondria in buffer A containing CCCP. The mitochondria were reisolated and washed. To analyze binding, the mitochondrial pellet was resuspended in buffer A containing CCCP and left on ice (- chase). For the chase reaction the mitochondria were reenergized and chased at 25 °C for the indicated times in min. The chase reaction was stopped by addition of valinomycin to 0.1 µg/ml. Where indicated (+ Prot. K), surface-bound precursor was digested with proteinase K, followed by addition of PMSF to 1 mM. The mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the precursor added to the assay; p and m, precursor or mature form of a protein, respectively. B, the surface-bound precursor of SU9-DHFR contains a tightly folded DHFR domain. The in vitro translated precursor of SU9-DHFR was bound to deenergized mitochondria; the mitochondria were reisolated, washed, and either resuspended in buffer A containing CCCP (Bind) or reenergized and chased for 20 min at 25 °C (Chase). The chase reaction was stopped by addition of valinomycin to 0.1 µg/ml. To analyze the folding status of the bound precursor, the mitochondria containing the bound precursor were reisolated and treated with proteinase K, followed by inactivation of the protease with 1 mM PMSF (+PK). The supernatant (Sup) was precipitated with trichloroacetic acid(30) . All samples were analyzed by SDS-PAGE and fluorography. pSU9-DHFR and mSu9-DHFR, uncleaved (``precursor'') and cleaved (``mature'') form of a fusion protein containing the first 69 amino acids of the N. crassa F(1)F(0)-ATPase subunit 9 precursor fused to mouse DHFR; DHFR*, folded, protease-resistant DHFR domain.



The DHFR domain of artificial precursors containing DHFR as a passenger protein is tightly folded, even after synthesis in vitro, but must be at least partly unfolded during import into mitochondria (19) . In order to determine whether unfolding is mediated by the mitochondrial receptors, SU9-DHFR was bound to uncoupled mitochondria. The mitochondria were then reisolated and divided into three aliquots. The first aliquot was analyzed directly for bound precursor (Fig. 1B, Bind - PK). The second aliquot was chased and then treated with protease in order to verify that the precursor had bound productively (Fig. 1B, Chase + PK). The third aliquot was treated with protease without a chase in order to check for release of the protease-resistant DHFR ``core'' fragment that is generated only from folded DHFR(19) . As the bound precursor was quantitatively converted to this core fragment (DHFR*; Fig. 1B, Sup + PK), its DHFR domain must have remained folded, only the presequence being exposed to the protease.

Productive Binding of Some Precursors Is Highly Saltsensitive

To investigate the mechanism by which different precursors bind to the mitochondrial surface, we measured productive binding of the precursors of SU9-DHFR, ADHIII, hsp60, the Rieske Fe/S protein, and AAC at different salt concentrations. The precursors of hsp60 and SU9-DHFR have a positively charged amino-terminal matrix-targeting sequence; they belong to a class of precursors whose import is preferentially mediated by Mas20p and does not require extramitochondrial ATP. The ADP/ATP carrier lacks a positively charged presequence; its import is preferentially mediated by Mas37p/Mas70p and does require extramitochondrial ATP. The precursors to ADHIII and the Rieske Fe/S protein both have a typical matrix-targeting signal, and their import is partly dependent on extramitochondrial ATP and mediated by both receptor subcomplexes^1(9, 20) .

In the experiments summarized in Fig. 2, the different precursors were bound to deenergized mitochondria in the presence of varying salt concentrations. The mitochondria were then reisolated, washed, and chased in low-salt buffer. Different aliquots of the mitochondria were then analyzed for productively bound precursor as outlined in Fig. 1. Adding 50-200 mM NaCl to the binding buffer inhibited productive binding of pSU9-DHFR and phsp60 almost completely (Fig. 2, A and C), but had little effect on productive binding of AAC (Fig. 2, B and C). Productive binding of the precursors of ADHIII (Fig. 2, B and C) and the Rieske Fe/S protein (not shown) showed an intermediate salt sensitivity. Thus, addition of 100 mM NaCl lowers the binding of ADHIII 3-4-fold.


Figure 2: Productive binding of the precursors of Su9-DHFR and of hsp60 is highly salt-sensitive. The precursors of SU9-DHFR and hsp60 (A) and those of ADHIII and the ADP/ATP carrier (AAC) (B) were translated in vitro and incubated with deenergized mitochondria in buffer A containing the indicated concentrations of NaCl at 0 °C for 5 min. The mitochondria were reisolated and washed. They were then either resuspended in buffer A containing CCCP and kept on ice (- chase) or suspended in buffer A, reenergized, and chased at 25 °C for 20 min (+ chase). Surface-bound precursor was digested with proteinase K followed by addition of PMSF to 1 mM. The mitochondria were reisolated and analyzed by SDS-PAGE, fluorography, and densitometric quantification of the bands. 20% STD, 20% of the precursor added to each assay. p and m, precursor or mature form, respectively, of each protein. C, densitometric quantification of the results shown in A and B (+ chase). For calculating chase efficiencies, the amount of precursor chased in the absence of added NaCl was taken as 100%.



Complete inhibition of productive binding of pSU9-DHFR and phsp60 is also seen upon addition of 35 mM Na(2)SO(4) instead of 100 mM NaCl, suggesting that the extent of inhibition is dependent on the ionic strength of the binding buffer (data not shown). An earlier report had suggested that pSU9-DHFR forms little, if any, productive binding intermediate(18) . This result can now be explained by the fact that binding and chase had been measured in a buffer containing 80 mM KCl; according to our results, this salt concentration inhibits productive binding of pSU9-DHFR by more than 90%.

Import of SU9-DHFR Is Also Salt-sensitive

To confirm that productive binding to mitochondria represents a partial reaction of the authentic import process, we measured the efficiency of import of SU9-DHFR and AAC at different salt concentrations. Addition of 100 mM NaCl to the import buffer inhibited import of SU9-DHFR almost completely, but had no effect on the import of AAC (Fig. 3), suggesting that inhibition of productive binding to the mitochondrial surface results in inhibition of import.


Figure 3: Import of SU9-DHFR into mitochondria is also salt-sensitive. The precursors of SU9-DHFR and ADP/ATP carrier (AAC) were translated in vitro and incubated with fully energized mitochondria in the presence of different concentrations of NaCl at 15 °C for the indicated times (in minutes). Nonimported precursor was digested with proteinase K followed by addition of PMSF to 1 mM. The mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the precursor added to each assay. p and m, precursor or mature forms, respectively, of the respective protein.



Productive Binding of SU9-DHFR to Uncoupled Mitochondria Is Dependent on Mas20p, but Independent of Mas37p/Mas70p

To determine the contributions of the two receptor subcomplexes to productive binding of different precursors, we measured binding of pSU9-DHFR and AAC to wild-type mitochondria, to mitochondria lacking both Mas37p and Mas70p, and to Mas20-deficient mitochondria which were respiratory competent because their normal level of Mas22p had been restored by a suppressor mutation(9, 13) . Mitochondria lacking both Mas37p and Mas70p bound pSU9-DHFR almost as effectively as did wild-type mitochondria, and binding to both types of mitochondria was highly salt-sensitive. In contrast, the Mas37p/Mas70p-deficient mitochondria failed to bind the AAC precursor (Fig. 4, A and B). Mitochondria lacking Mas20p, but having normal levels of Mas22p, had a greatly reduced ability to bind SU9-DHFR productively, whereas they could bind AAC as efficiently as did wild-type mitochondria. The residual productive binding of SU9-DHFR to these mitochondria seemed to be less salt-sensitive than binding to wild-type mitochondria (Fig. 4C).


Figure 4: Productive binding of SU9-DHFR to the mitochondrial surface is predominantly mediated by Mas20p. The precursors of SU9-DHFR and the ADP/ATP carrier (AAC) were translated in vitro and incubated as outlined in the legend of Fig. 2with deenergized mitochondria from wild-type cells (A), from a mutant lacking both Mas37p and Mas70p (Delta70Delta37) (B), or Delta20 res from a mutant lacking Mas20p, but with normal levels of Mas22p (C). The samples were chased as described in the legend to Fig. 2A and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the amount of precursor added to the assay. p and m, precursor or mature forms, respectively, of each protein.



Productive binding of SU9-DHFR to yeast mitochondria is thus predominantly mediated by Mas20p and independent of Mas37p/Mas70p. The Mas20p contains a highly acidic cytosolic domain that could bind positively charged presequences(4) .

A Mitochondrial Presequence Peptide Inhibits Productive Binding of pSU9-DHFR to Mitochondria

If Mas20p/Mas22p recognizes the presequence of precursors, its receptor function should be inhibited by chemically synthesized presequence peptides. Such peptides have indeed been shown to inhibit the import of precursors into isolated mitochondria(21, 22, 23) , but the mechanism of inhibition has remained uncertain: the extent of inhibition differed with different precursors and uncoupling by the amphiphilic peptides was not always ruled out. We tested the effect of a chemically synthesized peptide corresponding to the amphiphilic presequence of hsp60 from rat liver mitochondria. As a negative control, we chose the peptide synB2 (Fig. 5A); this peptide has the same number of basic residues and approximately the same length as the hsp60 presequence peptide, but is hydrophilic and inactive as a mitochondrial-targeting signal when attached to a passenger protein (24, 25) . The precursors of SU9-DHFR and AAC were bound to uncoupled mitochondria in the presence of different concentrations of each peptide; the mitochondria were then reisolated, washed, reenergized, and chased, treated with protease to remove nonimported precursor, and analyzed by SDS-PAGE and fluorography. Productive binding of pSU9-DHFR was almost completely inhibited by 0.5 µM hsp60 presequence peptide, but was unaffected by synB2. In contrast, productive binding of AAC (which lacks a basic presequence) was only partially inhibited (Fig. 5, B and C). Because the presequence peptide was added only during the potential-independent binding step, and import of the prebound precursor was measured after the mitochondria had been reisolated and washed, it is unlikely that the inhibitory effects of the peptide observed here merely reflect uncoupling of the mitochondria. As binding of pSU9-DHFR is so exquisitely sensitive to the presequence peptide, we conclude that productive binding of this precursor is mediated only via its targeting sequence. By contrast, binding of AAC must occur through additional binding sites.


Figure 5: Productive binding of SU9-DHFR to the mitochondrial surface is specifically inhibited by the presequence peptide of mammalian hsp60. A, amino acid sequences of the chemically synthesized prepeptide of hsp60 from rat liver mitochondria (pre-hsp60) and of the basic hydrophilic peptide synB2. B, the in vitro translated precursors of SU9-DHFR (pSU9-DHFR) and of the ADP/ATP carrier (AAC) were bound to deenergized mitochondria in the presence of the indicated concentrations of the hsp60 prepeptide (pre-hsp60) or of the hydrophilic peptide synB2(24) . The mitochondria were reisolated, washed, and reenergized. Bound precursor was then chased into the mitochondria at 25 °C for 20 min. Nonimported precursor was digested with proteinase K followed by addition of 1 mM PMSF, and the mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the total precursor added to each assay. C, quantification of the results shown in B. For calculating chase efficiencies, the amount of precursor chased in the absence of added prepeptide was taken as 100%.



Productive Binding of a Chemically Pure Precursor Protein

To exclude the possibility that the salt-sensitive productive binding of pSU9-DHFR and hsp60 requires unknown factors present in the reticulocyte lysate, we measured the productive binding of highly purified mitochondrial chaperonin 10 (cpn10)(15) . cpn10 is a matrix protein with an NH(2)terminal basic and amphiphilic matrix-targeting signal, which is not cleaved upon import. To study productive binding of pure cpn10, we expressed the protein in E. coli, purified it to more than 90% homogeneity, radioiodinated it, added it to deenergized yeast mitochondria in the presence of different salt concentrations, and chased the prebound protein into the mitochondria. No reticulocyte lysate was added at any step. cpn10 bound productively to the mitochondrial surface and about 15-20% of the prebound cpn10 could be chased into the mitochondria (Fig. 6, A and B). Productive binding of cpn10 to the surface of yeast mitochondria was highly salt-sensitive, being almost completely inhibited upon addition of 100 mM NaCl to the binding buffer (Fig. 6, B and C). Thus, salt-sensitive productive binding is also seen with a pure precursor protein. This binding step must therefore reflect a direct interaction of the precursor with the receptor sites at the mitochondrial surface.


Figure 6: Productive binding of the chemically pure precursor of yeast mitochondrial chaperonin 10 to the mitochondrial surface is highly salt-sensitive. A, productive binding of purified chaperonin 10 (cpn10). Two-hundred ng of I-labeled chemically pure yeast mitochondrial cpn10 (approximately 4 times 10^8 cpm/mg of protein) were bound to deenergized mitochondria. The mitochondria were then subjected to a chase for the indicated times (in minutes), treated with proteinase K to digest nonimported precursor, and analyzed by SDS-PAGE and autoradiography. B, productive binding of purified cpn10 is highly salt-sensitive. Two-hundred ng of I-labeled cpn10 was bound to isolated mitochondria in buffer A containing the indicated concentration of NaCl for 5 min at 0 °C. The samples were then treated as described in Fig. 2and analyzed by SDS-PAGE, autoradiography, and densitometric quantification of the autoradiograms. C, quantification of the results shown in B. The amount of precursor chased in the absence of added NaCl is taken as 100%.



Productively Bound cpn10 Can Be Cross-linked to the Import Receptor Mas20p

Chemical cross-linking has been widely applied to identify components of the mitochondrial import machinery that are in contact with a translocation-arrested precursor(16, 26, 27) . We used this approach to identify the receptor(s) to which cpn10 is productively bound. I-Labeled purified cpn10 was bound to deenergized mitochondria as described in Fig. 6; the mitochondria were reisolated, treated with the homobifunctional cross-linker disuccinimidyl suberate, and solubilized with SDS; the extract was then subjected to immunoprecipitation with antibodies against Mas20p, Mas70p, or porin (an abundant outer membrane protein not involved in protein import); finally, an aliquot of the extract as well as the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Cross-linking generated a major 30-kDa band (*A in Fig. 7) as well as a few very faint minor bands. The minor bands were not further identified. The 30-kDa band was immunoprecipitated by antibody against Mas20p, but not by antibody against Mas70p or porin. (Fig. 7, lanes 3-11). The cross-linked product disappeared if the cross-linker was added after the chase, indicating that the cross-linking to Mas20p is specific for the productively bound precursor (Fig. 7, lane 5).


Figure 7: The productively bound chemically pure precursor of mitochondrial chaperonin 10 can be specifically cross-linked to the import receptor Mas20p. I-Labeled chemically pure precursor of yeast mitochondrial cpn10 (200 ng; 4 times 10^8 cpm/mg of protein) was bound to isolated mitochondria in CCCP-containing buffer A for 5 min at 0 °C. The mitochondria were reisolated, washed, and divided into two equal aliquots. The first aliquot was resuspended in buffer A containing CCCP (- chase); the second aliquot was chased at 25 °C for 20 min. Each of the two aliquots was then divided further into two parts; one part was treated for 30 min at 0 °C with 200 µM of the homobifunctional cross-linker disuccinimidyl suberate (DSS), followed by addition of Tris/Cl (pH 7.4) to 0.2 M. The other part was left untreated. All four mitochondrial samples were then reisolated and either analyzed directly by SDS-PAGE and autoradiography or subjected to immunoprecipitation (I.P.) with antisera monospecific for either Mas20p, Mas70p, or porin followed by SDS-PAGE and autoradiography. M.W., protein molecular mass standards. *A, cross-linked product.



The productively bound precursor of mitochondrial cpn10 is thus directly bound to the receptor subunit Mas20p. As cpn10 is bound via its NH(2)-terminal targeting sequence (see above), this result strongly suggests that mitochondrial targeting sequences are recognized by the acidic Mas20p/Mas22p receptor subcomplex.


DISCUSSION

The present study shows that the basic and amphiphilic targeting sequence at the NH(2) terminus of a mitochondrial precursor protein is sufficient for recognition of the precursor by the acidic mitochondrial import receptor Mas20p. This result directly correlates a property of a mitochondrial targeting sequence with a component of the mitochondrial protein import machinery. The cytosolically exposed acid bristles of Mas20p and its partner subunit Mas22p are uniquely suited to bind the exposed basic mitochondrial targeting signals. This interaction is highly salt-sensitive and thus is largely mediated by electrostatic interactions. However, amphiphilicity of the targeting signal must also be important, because precursor binding is not inhibited by a basic peptide which is not amphiphilic.

The cross-linking data and the binding experiments with Mas20p-deficient mitochondria show that precursors with an NH(2)-terminal presequence are recognized by Mas20p, but these data do not exclude the possibility that presequences are also recognized by other receptor subunits. An additional subunit that probably recognizes presequences is Mas22p, which is tightly bound to Mas20p.^2

The identification of Mas20p as a ``presequence receptor'' does not imply a hierarchy of receptor function nor does it suggest that Mas20p is a ``master receptor''. On the contrary, our present data strengthen the view that the mitochondrial receptor subunits cooperate with one other as a functional unit in which the different subunits recognize different parts of a precursor protein (Fig. 8). For example, the precursors of ADHIII, the F(1)-ATPase beta-subunit, or the Rieske Fe/S protein probably bind simultaneously to several receptors: to Mas20p/Mas22p mainly via the presequence and to Mas37p/Mas70p mainly via the mature part. As a result, productive binding of these precursors is less sensitive to salt or presequence peptide.


Figure 8: Cooperation of receptor subunits in binding precursors to the yeast mitochondrial surface. Precursor binding is mediated by at least four distinct receptor subunits. These subunits exist as two hetero-oligomeric complexes that loosely interact with one another. Mas20p (possibly in association with Mas22p) binds mitochondrial precursor proteins through electrostatic interactions with the positively charged presequence, whereas the Mas70p/Mas37p receptor subcomplex interacts with the mature domain of the precursor molecule. OM, mitochondrial outer membrane; + and -, positively and negatively charged amino acids; dots between the soluble domains of Mas20p and Mas70p indicate a weak interaction between these receptor domains as suggested by studies using the two hybrid system.^5



Although Mas20p is a presequence receptor, several presequence-containing precursors are efficiently recognized by Mas37p/Mas70p. However, Mas37p/Mas70p differs from Mas20p in that it can only recognize precursors efficiently if it interacts with the mature part of the precursor. As Mas37p/Mas70p preferentially recognizes precursors that are bound to ATP-requiring cytosolic chaperones, it may also interact with these chaperones. A promising partner for such an interaction is the mitochondrial import stimulating factor MSF; MSF is an ATPase which prevents aggregation of mitochondrial precursors by binding them in an incompletely folded conformation(28) .

A complex containing at least three of the four known receptor subunits shown in Fig. 8can be isolated from digitonin-solubilized mitochondria (29) ; its existence in vivo is also suggested by the observation that the cytosolic domains of Mas20p and Mas70p interact with each other in the two hybrid system.^5 This complex may ensure efficient and specific binding of mitochondrial precursor proteins by simultaneously recognizing different portions of a precursor molecule.


FOOTNOTES

*
This study was supported by grants from the Swiss National Science Foundation (3-26189.89) and the Human Capital and Mobility Program of the European Economic Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Boehringer Ingelheim Foundation.

Supported by a long term fellowship from the Human Frontiers Science Program Organization.

**
To whom correspondence should be addressed: Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41-61-267-21-50; Fax: 41-61-267-21-75.

(^1)
S. Gratzer, T. Lithgow, R. E. Bauer, E. Lamping, F. Paltauf, S. D. Kohlwein, V. Haucke, T. Junne, G. Schatz, and M. Horst, submitted for publication.

(^2)
M. Horst and W. Oppliger, manuscript in preparation.

(^3)
The abbreviations used are: DHFR, dihydrofolate reductase; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PAGE, polyacrylamide gel electrophoresis; AAC, ADP/ATP carrier; PMSF, phenylmethylsulfonyl fluoride.

(^4)
S. Rospert and G. Schatz, manuscript in preparation.

(^5)
V. Haucke, T. Lithgow, and G. Schatz, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. N. Hoogenraad and P. B. Hoj for the gift of the pre-hsp60 peptide; H. Brütsch, R. Looser, and K. Schmid for excellent technical assistance; M. Jäggi, L. Müller, and V. Grieder for help with the artwork; V. Zellweger for secretarial assistance and members of the Schatz' laboratory for critical reading of the manuscript.


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