From the Institut für Physiologische Chemie,
Physikalische Biochemie und Zellbiologie der Universität
München, Goethestra
e 33, 80336 München, Federal Republic
of Germany and the
Institut für Zytobiologie der
Philipps-Universität Marburg, Robert-Koch-Str. 5, 35033 Marburg,
Federal Republic of Germany
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ABSTRACT |
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Translocation of preproteins across the
mitochondrial outer membrane is mediated by the TOM complex. Our
previous studies led to the concept of two preprotein binding sites
acting in series, the surface-exposed cis site and the
trans site exposed to the intermembrane space. We report
here that preproteins are bound to the cis site in a labile
fashion even at low ionic strength, whereas intermediates arrested at
the trans site remained firmly bound at higher salt
concentration. The stability of the trans site intermediate
results from interactions of both the presequence and unfolded parts of
the mature part of the preprotein with the TOM complex. Binding to the
trans site proceeded at rates comparable with those of
unfolding of the mature domain and appeared to be kinetically limited
by the unfolding reaction. Efficient binding to the trans
site and unfolding were observed with both outer membrane vesicles and
intact mitochondria whose membrane potential, , was dissipated.
Upon re-establishing
, trans site-bound preprotein
resumed translocation into the matrix. The rates of unfolding and
binding to the trans site were the same as those for
translocation into intact energized mitochondria. We conclude that
preprotein unfolding in intact mitochondria can take place without the
involvement of the translocation machinery of the inner membrane and,
in particular, the matrix Hsp70 chaperone. Further, preprotein
unfolding at the outer membrane can be a rate-limiting step for
formation of the trans site intermediate and for the entire
translocation reaction.
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INTRODUCTION |
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The transport of proteins across biological membranes involves the assistance of specific multi-subunit translocases (for reviews, see Refs. 1-7). Many of their subunits have been identified, but the detailed molecular events leading to the transfer of the proteins across the membranes are poorly understood. In some membrane systems, protein transport requires the unfolding of the translocating polypeptide chain. In mitochondria, targeting and translocation of a number of preproteins depends on cytosolic chaperones which keep the preproteins in a loosely folded, import-competent conformation (reviewed in Ref. 8). Some mitochondrial preproteins contain folded domains and must unfold upon their interaction with the translocation machinery. It has been suggested that the mitochondrial Hsp70 (mtHsp70)1 chaperone in conjunction with the translocation machinery of the inner membrane assists this unfolding reaction (9-13). According to one proposal, mtHsp70 participates in this process by actively "pulling" on the membrane-spanning polypeptide chain. A conformational change of mtHsp70 during the association with the preprotein is thought to be transduced into a directional force leading to the unfolding of domains still outside the mitochondrion (14, 15). In another view, mtHsp70 acts as part of a "molecular ratchet" which prevents the retrograde movement of the incoming polypeptide chain (16). Unfolding in this case is essentially due to the spontaneous breathing of the folded domain and is coupled to the reversible movement of the unfolded polypeptide chain across the membranes and trapping in the matrix space by stable binding to mtHsp70.
Using biochemical and genetic techniques, the TOM complex of the mitochondrial outer membrane has been shown to mediate specific recognition, unfolding, insertion, and translocation of preproteins (reviewed in Refs. 17 and 18). This complex contains preprotein receptors providing sites of initial interaction at the mitochondrial surface (e.g. see Refs. 19-21) and membrane-embedded components, which appear to form the translocation pore and facilitate membrane passage (22-24). Studies using intact mitochondria and isolated outer membrane vesicles (OMV) have provided a coarse picture of how preproteins containing N-terminal targeting signals (presequences) are translocated. At the mitochondrial surface, presequences are specifically recognized by the co-operative action of the receptors Tom20/Tom22 which form a presequence recognition site termed cis site (25, 26). At this site, the preprotein is bound mainly through electrostatic interactions. Translocation of the preprotein is initiated by the transfer of its presequence across the outer membrane and binding at the so-called trans site (27). The presequence of a trans site-bound preprotein is closely associated with the membrane-embedded component Tom40 (22) and is exposed to the intermembrane space (27). Preprotein binding at the trans site is accompanied by unfolding of adjacent folded domains, a prerequisite for protein import into mitochondria (9, 28).
Essential features of preprotein translocation across the outer membrane, preprotein unfolding, and binding at the trans site are poorly understood. The rates of binding to the trans site and the contribution of mature domains to the interaction have not been analyzed. Further, the physiological relevance of the unfolding activity of the outer membrane is unclear. The function of the trans site as an intermediate stage in preprotein translocation has not been established in intact mitochondria.
Here, we present a biochemical characterization of the interaction of preproteins with the trans site, and we demonstrate the importance of this site as an intermediate state of translocation into intact mitochondria. Further, we present evidence that unfolding can occur at the stage of outer membrane translocation, i.e. before a contact between the preprotein and mtHsp70 chaperone is established. The unfolding event at the outer membrane was found to be rate-limiting for both the interaction of the preprotein with the trans site and for the entire translocation process.
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MATERIALS AND METHODS |
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Biochemical Procedures-- The following published procedures were used: growth of Neurospora crassa wild-type strain 74A and purification of mitochondria and mitochondrial OMV (29); treatment of OMV with trypsin, raising antisera, and purification of immunoglobulin G (IgG; Ref. 30), with the modification that IgGs were concentrated by ultrafiltration in Centriprep tubes (Amicon); transcription and translation reactions in reticulocyte lysate using [35S]methionine (ICN Radiochemicals) as radioactive label (31); preparation of both subunits of N. crassa matrix processing peptidase (MPP); and cleavage of the presequence by MPP (27, 32). Co-immunoprecipitation of the TOM complex with bound translocation intermediates was performed as described earlier using antibodies directed against various TOM complex components (33).
Preprotein Synthesis in Reticulocyte Lysates-- The following fusion proteins were synthesized in reticulocyte lysate: pCyt c1-DHFR containing the presequence (amino acid residues 1-34) of N. crassa cytochrome c1 in front of mouse dihydrofolate reductase (DHFR; Ref. 34); and pSu9-DHFR containing the first 69 amino acid residues of N. crassa subunit 9 of the mitochondrial F0-ATPase (pSu9) in front of DHFR (35). Construction of a truncated version of pSu9-DHFR termed pSu9(+7) was described earlier (22). This preprotein contains only 7 amino acid residues after the first 69 residues of pSu9 and was synthesized by run-off translation (22). The preprotein pSu9-DHFRmut harbors mutations in the DHFR domain, resulting in impaired folding (36). Urea-denatured pSu9-DHFR (termed pSu9-DHFRurea) was prepared by precipitating reticulocyte lysate containing pSu9-DHFR with ammonium sulfate (66% saturated solution). After centrifugation for 15 min at 15,000 × g, the precipitate was dissolved in 10 mM MOPS-KOH, pH 7.2, containing 8 M urea in the same volume as the lysate input.
Binding and Import of Preproteins in Vitro-- OMV were suspended in 100 µl of import buffer A (0.2 mg/ml bovine serum albumin, 2.5 mM MgCl2, 10 mM KCl, and 15 mM MOPS-KOH, pH 7.2) in the absence or presence of 1 mM NADPH and 1 µM methotrexate (MTX; Refs. 25 and 27). Import into freshly isolated mitochondria was performed in import buffer B (buffer A supplemented with 220 mM sucrose). To avoid translocation of the presequence across the inner membrane, 20 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to dissipate the membrane potential. OMV or mitochondria were incubated with reticulocyte lysate containing the radiolabeled preproteins for the desired times at various temperatures. Samples were diluted with 700 µl of high salt (HS) or low salt (LS) buffers (10 mM MOPS-KOH, 1 mM EDTA, pH 7.2, and 120 or 20 mM KCl, respectively) containing 220 mM sucrose for experiments with mitochondria. For the analysis of binding, OMV or mitochondria were reisolated by centrifugation for 20 min at 125,000 × g or 10 min at 12,000 × g, respectively. To assay for preprotein import into mitochondria, proteinase K (50 µg/ml) was added and halted after 15 min at 0 °C by 1 mM phenylmethylsulfonyl fluoride. After addition of 1 ml of SEM buffer (220 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2) and reisolation of the organelles, pellets were subjected to SDS-PAGE (29). Imported proteins were visualized by fluorography and quantitated by phosphoimager analysis (FUJI X BAS 1500).
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RESULTS |
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Conditions were established under which preproteins were bound exclusively to either the cis or trans sites. We employed fusion proteins termed pSu9-DHFR or pCyt c1-DHFR comprised of the presequences of subunit 9 of the F0-ATPase or of cytochrome c1, respectively, and mouse DHFR. The preproteins were bound at 25 °C to purified mitochondrial outer membrane vesicles (OMV) in the presence or absence of MTX and NADPH. Addition of these compounds prevents the unfolding of the DHFR moiety and thus precludes preproteins from associating with the trans site (27). In the presence of MTX/NADPH, the preproteins remained bound exclusively at the surface-exposed cis site in a salt-sensitive fashion. Bound preprotein was completely released from the OMV upon treatment with buffer containing a high concentration of KCl (Fig. 1A). In contrast, when incubated in the absence of MTX/NADPH, the preprotein remained bound to OMV even in buffers of high ionic strength (Fig. 1B). Even after treatment with 600 mM KCl, only 40% of the bound preprotein was released from the OMV (not shown), indicating that ionic interactions play a minor role for the association with the trans site. Binding to both cis and trans sites was largely dependent on the function of the surface receptors which can be degraded by treatment of the OMV with trypsin (25). We conclude from these data that we can distinguish between preprotein binding to the cis and trans sites of the mitochondrial outer membrane on the basis of their differential sensitivity to salt extraction. Binding in the presence or absence of MTX/NADPH combined with a treatment at low or high salt concentrations allows the exclusive occupation of either cis or trans sites.
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Using these criteria to discriminate between cis- and trans-site-bound preproteins, we investigated the time courses of preprotein binding to these two sites. At 25 °C, the association of pSu9-DHFR with the cis site occurred within seconds, whereas only slow binding with a half time of 5 min was observed for the trans site (Fig. 2A). Binding to the trans site was significantly slowed down after removal of the surface receptors by trypsin treatment. When preprotein binding to untreated OMV was performed at 0 °C instead of 25 °C, association with the cis site still took place rapidly, but negligible amounts of preprotein were bound to OMV in a salt-resistant fashion (Fig. 2B), i.e. no association with the trans site was detectable under these conditions.
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To analyze the time course of unfolding of the DHFR domain in these binding experiments, samples were treated with protease under conditions which leave folded DHFR intact, but completely degrade unfolded DHFR. During incubation at 25 °C in the absence of MTX/NADPH, unfolding occurred at a rate similar to that of trans site binding (cf. Fig. 2, B and C). No unfolding was observed at 0 °C, consistent with the finding that no salt-resistant state was reached under this condition. Thus, stable binding of pSu9-DHFR to the trans site of OMV is accompanied by the simultaneous unfolding of the DHFR domain. Binding to the trans site and/or unfolding of the mature part of the preprotein require incubation at higher temperature.
Can the trans site be reached after binding to the cis site? pSu9-DHFR was pre-bound at the cis site either by incubation in the presence of MTX or at 0 °C, and unbound material was removed by centrifugation. When OMV carrying cis site-bound pSu9-DHFR were incubated at 25 °C in the absence of MTX, a significant fraction of the preprotein was translocated to the trans site as indicated by the resistance to treatment at increased ionic strength (Fig. 2D). No translocation to the trans site was observed when the OMV were left on ice or were incubated in the presence of MTX. These data show that the cis site is used as an intermediate stage during preprotein translocation to the trans site of the outer membrane.
The lack of salt-resistant binding at 0 °C either could be due to the slow unfolding of the mature domain precluding translocation of the presequence across the outer membrane, or could result from a slow association of the preprotein with the trans site. In the first case, the rates of salt-resistant binding are expected to increase, if the preprotein harbors an unfolded DHFR domain. To test this experimentally, the preprotein pSu9-DHFR in the native form or after denaturation in 8 M urea (termed pSu9-DHFRurea) were incubated with OMV for various times at 15 °C and 0 °C. Then, OMV were treated with high salt buffer, reisolated and analyzed for bound preprotein. pSu9-DHFR became associated with OMV in a salt-resistant fashion at 15 °C but not at 0 °C (Fig. 3). In comparison, higher amounts of pSu9-DHFRurea were bound at both temperatures, i.e. the rates of salt-resistant binding of pSu9-DHFRurea were considerably higher than those of folded pSu9-DHFR. Thus, temperature-dependent unfolding of the mature domain determines how fast the presequence can be translocated across the outer membrane and can associate with the trans site. Treatment with urea circumvents this temperature dependence, demonstrating that presequence binding at the trans site is not a rate-limiting event.
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To investigate whether the mature part of the preprotein contributes to salt-resistant binding at the trans site, we used a truncated preprotein (termed pSu9(+7); Ref. 22) consisting of the presequence of Su9 and only seven additional amino acid residues. Its binding under low and high salt conditions was compared with that of pSu9-DHFR. After incubation at 0 or 25 °C and treatment with low salt buffer, a large fraction of the added pSu9(+7) was bound to the OMV (Fig. 4A). Following treatment with high salt buffer, only minor amounts of pSu9(+7) remained associated with the OMV. This was in contrast to pSu9-DHFR, which bound in a salt-resistant fashion at 25 °C. These data suggest that the mature part of the preprotein contributes to the stable binding at the trans site. A similar conclusion can be drawn from binding studies with pSu9-DHFRmut (carrying mutations in the DHFR domain which impair stable folding; Ref. 36) and pSu9-DHFRurea. Both preproteins associated with OMV in a salt-resistant fashion, even when binding was performed at 0 °C (Fig. 4B). In conclusion, preproteins can reach the salt-resistant binding state without the need for an increased temperature provided translocation is not hindered by a folded mature domain following the presequence. Unfolded segments of the mature portion of the polypeptide chain contribute to stable interaction with the translocation machinery of the outer membrane. Receptors are unlikely to play a major role in this interaction. The low amounts of pSu9-DHFR (Figs. 1B and 2A), pSu9-DHFRmut, and pSu9-DHFRurea (not shown) bound in the absence of receptors were associated in a salt-resistant fashion as was preprotein bound in the presence of receptors.
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Do the reactions of presequence translocation and of concomitant
unfolding of the mature portion of the preprotein observed with
isolated outer membranes reliably reflect the import process in intact
mitochondria? We first followed the unfolding of the DHFR domain during
the incubation of pSu9-DHFR with isolated mitochondria, and we
monitored the sequestration of the presequence cleavage site into the
translocation channel. To uncouple translocation across the outer
membrane from that across the inner membrane, isolated mitochondria
were used whose membrane potential, , was depleted. Upon
incubation of these mitochondria with pSu9-DHFR at 25 °C, the
preprotein was bound rapidly, but the presequence was not cleaved by
endogenous MPP, indicating that it had not been transferred across the
inner membrane (Fig. 5, left
panels). Nevertheless, the mature part of the preprotein became
unfolded with a half time of less than 5 min (middle
panels). The presequence cleavage site became inaccessible to
externally added, purified MPP at virtually the same rate (right
panels), indicating insertion of the presequence part of the
preprotein into the outer membrane. Neither unfolding nor presequence
sequestration was seen at 0 °C (not shown). Taken together, intact
mitochondria can translocate the presequence of a preprotein across the
outer membrane, and concomitantly the mature part of the preprotein
unfolds. Both reactions occur independently of the further transfer of
the polypeptide chain across the inner membrane.
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We next asked if preprotein bound to the trans site
represents a faithful intermediate of translocation into mitochondria. This has been questioned recently (37). The preprotein
pSu9-DHFRurea was first bound to mitochondria which were
uncoupled by the addition of CCCP. After removal of free or
cis site-bound material by reisolation of the mitochondria
in high salt buffer, the membrane potential, , was reestablished
by the addition of dithiothreitol which quenches CCCP. A second
incubation was performed to permit further transport of
trans site-bound preprotein into the matrix. A significant fraction of this preprotein acquired protection against proteinase K
and its presequence was processed (Fig.
6, lanes 3 and 4)
similar to what was observed for the import of preprotein freshly added before the second incubation (lanes 7 and 8). In
contrast, hardly any proteinase K-resistant preprotein was detected
when
was lacking in both incubations (lanes 1 and
2 and 5 and 6). These data indicate
that preprotein transiently arrested at the trans site
resumed its journey into the matrix. Thus, the trans binding stage can be considered a productive intermediate of the overall translocation process.
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To prove that pSu9-DHFR bound to uncoupled mitochondria in a salt-resistant fashion was in intimate contact to the translocation machinery of the outer membrane, the TOM complex was isolated by immunoprecipitation using antibodies against Tom20, Tom22, and Tom40 and analyzed for bound preprotein. A significant fraction of added pSu9-DHFR was co-immunoprecipitated with the TOM complex after preprotein binding under conditions leading to occupation of the trans site (Fig. 7). When the preprotein was arrested at the cis site, no such stable association with the TOM complex was seen, even when co-immunoprecipitation was performed in the presence of low salt concentrations. Essentially the same was observed with isolated OMV (not shown; 22). These data demonstrate that the preprotein, after inserting its presequence into the outer membrane of uncoupled mitochondria is stably associated at the trans site of the TOM complex, whereas at the cis site, it is bound in a labile fashion. In summary, the results obtained with intact mitochondria are fully consistent with the conclusions drawn from the partial reactions detected with OMV (Ref. 27; see above). Preprotein bound at the trans site represents a functional intermediate in the translocation pathway from the cytosol into the mitochondrial matrix.
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Unfolding of the mature domain at the mitochondrial outer membrane is rate-limiting for the initiation of translocation (see above, Fig. 3). To analyze whether unfolding also determines the rate of the overall translocation into the matrix, we compared the time courses of unfolding of pSu9-DHFR upon incubation with uncoupled mitochondria (by the addition of CCCP) and of translocation into the matrix space of energized mitochondria. Virtually identical rates were found for these two reactions, when they were performed at 25 °C in parallel experiments (Fig. 8A). No import was observed with uncoupled mitochondria. The similarity of the rates of unfolding and translocation suggests unfolding to represent the rate-limiting step for translocation of pSu9-DHFR. This conclusion was supported by a translocation experiment with intact mitochondria and preproteins harboring unfolded DHFR domains. While there was essentially no import of folded pSu9-DHFR at 0 °C, pSu9-DHFRmut and pSu9-DHFRurea became efficiently translocated into mitochondria under these conditions (Fig. 8B). Apparently, a preprotein can translocate into mitochondria at 0 °C provided its mature domain is unfolded (see also Ref. 38). In conclusion, unfolding of the mature domain of pSu9-DHFR is the rate-limiting step not only for association with the trans site but also for translocation into the matrix. The reason for the inability of the folded preprotein to translocate at 0 °C seems to be the lack of efficient unfolding of the DHFR domain at the outer membrane rather than the membrane passage. Folding of the preprotein precludes interaction with the trans site of the outer membrane and further transfer to and across the translocation machinery of the inner membrane.
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DISCUSSION |
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The present contribution provides a biochemical characterization of the trans site of the TOM complex. This preprotein binding site can be distinguished from the cis site by several criteria. First, when a preprotein is present at the cis site, its presequence can be cleaved off by mitochondrial processing peptidase (MPP) added to the outer face but not to the inner face of OMV. When the preprotein is bound at the trans site, on the other hand, its presequence cannot be cleaved by MPP added on the cytosolic side; rather, it is exposed on the intermembrane space side of the outer membrane and can be processed by MPP enclosed in the lumen of OMV (27). Second, preproteins associated with the trans site are bound in a salt-resistant and stable fashion, whereas preproteins associated with the cis site are rapidly released, especially at higher ionic strength. Presumably, the interaction with the cis site is predominantly of electrostatic nature (25, 26). Third, binding at the cis site occurs mainly via the presequence (25), whereas both the presequence and segments of the mature part of the preprotein are required for stable association with the trans site. Apparently, different modes of interaction are operating at the two preprotein binding sites. Fourth, the presequence is bound at the cis site by Tom20 and Tom22 (22, 25). At the trans site, the presequence has left the vicinity of the former two components and is bound mainly to Tom40, a membrane-embedded component (22). The contact between the presequence and Tom40 is established early in the translocation reaction, when the preprotein is still bound to Tom20-Tom22 at the mitochondrial surface. Therefore, Tom40 appears to play an important role in guiding the presequence into and across the translocation channel. One of the small subunits of the TOM complex, Tom5, also seems to participate in the transfer of the preprotein from the mitochondrial surface into and across the outer membrane (24). In conclusion, the cis and trans sites of the mitochondrial outer membrane translocase represent distinct preprotein binding states that become occupied in a sequential manner during preprotein transport.
The presequence is essential for the entry of the preprotein into the translocation channel and for association with the trans site (27). Our study shows that parts of the mature domain following the presequence appear to interact with the translocation machinery and contribute to the overall binding stability at the trans site. The binding of this unfolded mature portion to the TOM complex is reversible. The mature protein was found to be released from the import machinery in a retrograde translocation reaction, when the presequence of a trans-site bound preprotein was enzymatically removed by adding MPP at the intermembrane space side of the outer membrane (27). Thus, the combination of reversible contacts of both the presequence and the mature parts are responsible for the high stability of a trans-site bound preprotein. The precise nature of interaction of the preprotein with the trans site remains to be determined. At any rate, the interactions appear to be mediated mainly or even exclusively through the TOM complex since the association of the trans site-bound preprotein was maintained when the outer membrane was lysed in detergent solutions and the TOM complex was isolated by co-immunoprecipitation.
Unfolding of a domain such as DHFR immediately following the
presequence is a requirement for translocation of the presequence across the outer membrane. Only when the DHFR is unfolded does the
presequence cleavage site become exposed to the lumen of OMV (27). We
report here two important characteristics of the unfolding reaction.
First, efficient unfolding was seen in intact uncoupled mitochondria.
In the absence of a membrane potential, , the presequence cannot
cross the inner membrane (39). Therefore, the unfolding observed in
intact uncoupled mitochondria occurred at the stage of outer membrane
interaction, i.e. before the preprotein made contact with
components of the TIM complex, in particular Tim44, and with mtHsp70
chaperone in the matrix (40-43). Thus, at least in the experimental
system used in this study, mtHsp70 does not play an essential role in
the unfolding of preproteins at the mitochondrial surface (see Ref.
15).
Second, unfolding at the mitochondrial surface proceeded at rates indistinguishable from those of the entire translocation process. This argues for unfolding at the mitochondrial outer membrane being a rate-limiting step for the overall translocation process, at least in the experimental system used. This interpretation must a priori apply to preproteins with presequences so short that they cannot reach the inner membrane or matrix space components without preceding unfolding of the mature domains following the presequence part. In the case of longer presequences or presequences followed by stretches of unfolded mature portions, participation of the translocation machinery of the inner membrane, presumably of Hsp70, has been reported recently (15). Also in these cases, at least partial unfolding may occur at the stage of outer membrane interaction.
In intact energized mitochondria the N-terminal segments of longer presequences are rapidly passed on from the outer membrane to the TIM complex and subsequently to mtHsp70 in the matrix space. Binding of the preprotein to components of the TIM complex (44, 45) and to mtHsp70 prevents the reversible retrograde sliding back out of the translocation channel (16) and, thus, performs a similar function as does the reversible interaction with the trans site. Also from a kinetic point of view, our findings with pSu9-DHFR do not support an active role for mtHsp70 in the unfolding reaction, as unfolding at the stage of outer membrane interaction takes place at rates comparable with those measured for the entire translocation reaction. Any assistance in the unfolding process, however, should accelerate the rate of translocation, an observation we did not make (see Ref. 15). Thus, our data are compatible with the molecular or Brownian ratchet model. In such a minimal model, net movement of the translocating polypeptide chain across the membranes is achieved by successive binding of multiple molecules of mtHsp70, which prevent the reverse movement of the preprotein out of the translocation channel. Taken together, our data with pSu9-DHFR suggest that mtHsp70 is not required to actively facilitate unfolding but rather may act passively by trapping those segments of the polypeptide chain that are newly imported into the matrix space.
There is no external energy requirement such as ATP hydrolysis for
preprotein binding to the trans site and for the concomitant unfolding reaction (27). Since unfolding of the mature domain of
pSu9-DHFR is a prerequisite for stable preprotein binding at the
trans site, unfolding must occur in a spontaneous fashion. N-terminal segments of the preprotein may then slide into the translocation channel and associate with the trans site. The
energy gained from interactions at the trans site seems to
be sufficient to compensate for the loss of free energy of folding,
thus leading to a shift in the equilibrium toward net unfolding. In
this respect, the function of the trans site can be compared
with that of a molecular chaperone; binding to spontaneously unfolded
polypeptide segments shifts the equilibrium of folded and unfolded
states to the latter. Notably, binding to the TOM complex was
previously found to prevent aggregation of a preprotein in
transit, reflecting a chaperone-like activity (27). This property
of the TOM complex may be mechanistically useful for keeping unfolded
preproteins in an import-competent conformation and may be important
also for other preprotein translocases. Further movement of the
preprotein from the trans site to the inner membrane is
likely to be mediated by interaction with Tim23 (46). The requirement
for at this stage may represent the energy input for relieving
the tight interaction with the trans site.
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ACKNOWLEDGEMENTS |
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We thank Dr. B. Westermann for providing the plasmid encoding the pSu9(+7) protein, P. Heckmeyer and M. Braun for excellent technical assistance, and Dr. M. Harmey for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants of the Sonderforschungsbereich 184 of Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and by fellowships of the European Molecular Biology Organization (to D. R.) and the Boehringer Ingelheim Fonds (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this paper.
¶ Present address: Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tübingen, Federal Republic of Germany.
** To whom correspondence should be addressed. Tel.: 49-6421-28-6449; Fax: 49-6421-28-6414; E-mail: Lill{at}mailer.uni-marburg.de.
1 The abbreviations used are: mtHsp70, mitochondrial Hsp70 chaperone; OMV, outer membrane vesicles; MPP, matrix processing peptidase; DHFR, dihydrofolate reductase; MOPS, 4-morpholinepropanesulfonic acid; MTX, methotrexate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; HS, high salt; LS, low salt; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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