(Received for publication, April 15, 1997)
From the Institut für Physiologische Chemie,
Physikalische Biochemie und Zellbiologie der Universität
München, Goethestraße 33, 80336 München, Federal Republic
of Germany and § Institut für Zytobiologie der
Philipps-Universität Marburg, Robert-Koch-Str. 5, 35033 Marburg,
Federal Republic of Germany
During preprotein transport across the mitochondrial outer membrane, the N-terminal presequence initially binds to a surface-exposed site, termed cis site, of the protein translocation complex of this membrane (the TOM complex). The presequence then moves into the translocation pore and becomes exposed at the intermembrane space side. Membrane passage is driven by specific interaction of the presequence with the trans site. We have used chemical cross-linking to identify components in the vicinity of the translocating presequence. Preproteins bound to the surface-exposed cis site can be cross-linked via their N-terminal presequence to Tom20 and Tom22, demonstrating their direct association with this part of the preprotein. In addition, the presequence establishes an early contact to Tom40, a membrane-embedded protein of the TOM complex. Upon further entry of the preprotein into the translocation pore, the presequence loses its contact with Tom20/Tom22, but remains in firm association with Tom40. Our study suggests that Tom40 plays an important function in guiding the presequence of a preprotein across the mitochondrial outer membrane. We propose that Tom40 forms a major part of the trans presequence binding site.
In most nucleus-encoded mitochondrial proteins, the targeting signal is contained in an N-terminal extension, the mitochondrial targeting sequence (presequence). The presequences are recognized by the mitochondrial outer membrane translocation system (TOM complex)1 and the translocation apparatus of the inner membrane (TIM complex). Both complexes can act in a coordinated fashion to facilitate the insertion of preproteins into these two membranes as well as the translocation into the internal compartments, the intermembrane space and the matrix (for reviews, see Refs. 1-6).
Investigations during the last decade have identified many components participating in mitochondrial protein import and have provided us with a coarse picture of the molecular mechanism of the transport process. At the mitochondrial outer membrane, import is initiated through a series of reactions, including preprotein recognition, unfolding, insertion, and translocation (reviewed in Ref. 5). These processes are facilitated by the multisubunit TOM complex. Members of the TOM complex can be grouped into components that are protease-sensitive and act as preprotein receptors and components that are more embedded into the membrane and that may form a translocation pore. The central receptor components are Tom20 and Tom22. They form a receptor unit that is essential for the formation of a presequence binding site defined as the "cis site" (7, 8). Ionic interactions with the presequence appear to be the dominant force of preprotein recognition at this site, as bound preproteins readily dissociate at higher ionic strength (8-11). The other receptors, Tom37, Tom70, and possibly Tom71, form a subcomplex and cooperate in the import of a subset of preproteins (12, 13). From this latter receptor unit, preproteins are passed on to Tom20/Tom22, a process which may be facilitated by the interaction of Tom20 and Tom70 (14).
From the receptors, preproteins move into and across a putative translocation pore, which may be formed by the membrane-embedded components of the TOM complex (7). These components include the essential protein Tom40 (15, 16) and three small Tom proteins (Tom5, Tom6, and Tom7) (17-19). The transfer of the presequence into the translocation pore is accompanied by the concomitant unfolding of the mature part of the preprotein and results in the formation of a stably bound translocation intermediate in which the presequence is bound to a specific binding site termed "trans site." In this binding state, the presequence is exposed to the intermembrane space (7). The questions of how this passage across the outer membrane occurs and which components are involved in forming the trans site are still unresolved.
The aim of the present study is to further unravel the mechanistic events of the membrane translocation process across the mitochondrial outer membrane. Furthermore, we attempted to identify those components that are in close contact to the translocating preprotein and that serve as specific binding partners for the presequence. For this purpose, we identified the nearest neighbors of the translocating polypeptide chain by chemical cross-linking. The preprotein was arrested in distinct translocation intermediate states that were established in either purified outer membrane vesicles (OMV) (20) or in intact mitochondria. Particular emphasis was on the identification of the cross-linking partners of the presequence part of the preprotein to gain insight into the molecular nature of the presequence-specific trans site.
The results presented here demonstrate that, in addition to the well known presequence binding partners Tom20 and Tom22, Tom40 is in close contact with the presequence part of a translocating polypeptide chain. This intimate contact is established early on in the translocation event, when the preprotein is still bound to the surface-exposed cis site. Upon entering the translocation pore, the presequence looses its contact to the cis site-forming proteins Tom20/Tom22, but remains bound in the close vicinity of Tom40. Our data suggest that Tom40 plays an important role in presequence interaction during the passage of preproteins across the mitochondrial outer membrane.
The following published procedures were used: growth of Neurospora crassa wild type strain 74A and purification of mitochondria and mitochondrial OMV (20); treatment of OMV with trypsin, raising antisera, and purification of IgG (21), with the alteration that IgG were concentrated by ultrafiltration in Centriprep tubes (Amicon); transcription and translation reactions in reticulocyte lysate using [35S]methionine (ICN Radiochemicals) as radioactive label (22); analysis of proteins by SDS-polyacrylamide gel electrophoresis (PAGE) and fluorography of the resulting gels (23); blotting of proteins onto nitrocellulose and immunostaining of blotted proteins using the ECL chemiluminesence detection system (Amersham Corp.) (20). The amount of radioactive protein was quantitated on a PhosphorImager FUJI X BAS 1500. Protein concentrations were determined by the Coomassie dye-binding assay using IgG as a standard (Bio-Rad).
Preprotein Synthesis in Reticulocyte LysatesThe following fusion proteins were synthesized in reticulocyte lysate: PreSu9-dihydrofolate reductase (DHFR) containing the first 69 amino acids of subunit 9 of the mitochondrial F0-ATPase (pSu9) in front of mouse DHFR; this protein was termed pSu9-DHFR and contains cleavage sites for matrix processing peptidase after residues 35 and 66 (24). A truncated version of pSu9-DHFR termed pSu9-DHFR13 was generated by cleaving the plasmid pGEM4 harboring the coding region of pSu9-DHFR at unique site with AccB7I restriction enzyme before transcription. This results in a linearized DNA containing the coding region for amino acid residues 1-69 of pSu9 plus the first 13 residues of DHFR. A protein termed pSu9(+7) containing amino acid residues 1-69 of pSu9 plus 7 additional residues was generated by linearizing plasmid pGEM3 harboring the coding region for this sequence with EcoRI (plasmid kindly provided by Dr. B. Westermann). All these truncated proteins were synthesized by run-off translation for 1 h at 25 °C in reticulocyte lysate. Puromycin (250 µM) was added at the end of the synthesis reaction, and incubation was continued for 3 min at 25 °C to release nascent protein from the ribosomes.
In Vitro Import of Preproteins, Cross-linking, and Co-immunoprecipitationFor import or binding experiments, all vials were coated with fatty acid-free bovine serum albumin (1 mg/ml, 15 min) before use to reduce unspecific interactions of precursor proteins with tube walls. OMV were suspended in import buffer (0.25 mg/ml bovine serum albumin, 20 mM KCl, 2.5 mM MgCl2, 10 mM MOPS/KOH, pH 7.2) in the absence or presence of 1 mM NADPH and 1 µM methotrexate (MTX). In experiments using mitochondria, the import buffer was supplemented with 220 mM sucrose, and with 30-45 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to dissipate the membrane potential across the inner membrane. Reticulocyte lysate containing the radiolabeled precursor proteins was then added and incubated with OMV or mitochondria for the desired times at various temperatures. Samples were diluted with high (HSW) or low salt (LSW) washing buffer (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. Finally, OMV or mitochondria were reisolated by centrifugation for 20 min at 125,000 × g or 10 min at 12,000 × g, respectively.
For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 µM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added. After 40 min at 0 °C, excess cross-linker was quenched by the addition of 100 mM Tris-HCl, pH 7.5, or 100 mM glycine, pH 8.0. Aliquots were removed before and after the addition of the cross-linking reagents, proteins were precipitated by trichloroacetic acid and analyzed by SDS-PAGE. For immunoprecipitation, samples were dissolved in lysis buffer (1% SDS, 0.5% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.2). After incubation for 5 min at 25 °C, the lysed material was diluted 40-fold with lysis buffer lacking SDS. After a clarifying spin (15 min at 20,000 × g), the supernatant was incubated with antibodies that were coupled to protein A-Sepharose beads. Co-immunoprecipitation of the TOM complex with bound translocation intermediates was performed as described earlier using antibodies directed against Tom40 (15).
To identify the components involved in forming the previously
defined cis and trans sites of the TOM complex
(7), a fusion protein comprised of the presequence part of subunit 9 of
the F0-ATPase and mouse DHFR termed pSu9-DHFR was used.
Cis site binding was established by incubating isolated OMV
with the fusion protein at 0 °C in the presence of 1 µM MTX followed by a wash of the OMV with LSW (20 mM KCl). Under this condition, the preprotein was loosely
bound to the OMV and dissociated at higher ionic strength (8) (see also
Fig. 1A, lanes Total).
Trans site interaction was established by performing the
binding reaction at 25 °C in the absence of MTX. In this case, the
majority of the preprotein was bound in a stable, salt-resistant
fashion. Residual cis site-bound material could be removed
by a wash with HSW (120 mM KCl), thus resulting in an
almost exclusive accumulation of trans site-bound preprotein. This latter material was firmly associated with the TOM
complex and could be isolated by co-immunoprecipitation using antibodies against, e.g. Tom40 (Fig. 1A), in
contrast to what was found for cis site-bound material.
Using these two binding conditions, we analyzed the molecular
environment of the bound preprotein by a cross-linking approach.
A preprotein bound to OMV under
cis and trans conditions can be cross-linked to
various TOM components. A, a preprotein can be
co-immunoprecipitated with antibodies against Tom40, only when bound to
the trans site. Radioactively labeled pSu9-DHFR was bound to
OMV for 20 min at 0 or 25 °C in the presence or absence of
MTX/NADPH, respectively. The reaction mixtures were chilled on ice and
diluted with LSW or HSW buffers (containing 20 or 120 mM
KCl, respectively). OMV together with cis or
trans site-bound material were reisolated (20 min,
125,000 × g) and resuspended in SEM buffer with or
without MTX/NADPH. Co-immunoprecipitation with antibodies against Tom40
or with antibodies derived from preimmune serum was performed. As a
control for the input, an aliquot (Total) representing 25%
of the material used for co-immunoprecipitation is shown. B,
pSu9-DHFR was bound to OMV under cis and trans
conditions and the OMV were washed as described in A. To the
indicated samples, 13 µM of F1 presequence peptide was added. The cross-linking reagent DSG
(final concentration 117 µM) was added, and samples were
incubated for 40 min at 0 °C. Aliquots were removed before (
DSG) and after the cross-linking reaction
(+DSG). After quenching of the cross-linker, proteins were
precipitated with trichloroacetic acid, and samples were analyzed for
radioactive pSu9-DHFR and its cross-linking products by SDS-PAGE and
fluorography. Apparent molecular masses are given on the left.
C, immunoprecipitation of cross-linking products. After performing
the binding and cross-linking reactions as described in B,
OMV were reisolated and solubilized with SDS- and Triton
X-100-containing buffer. Aliquots were subjected to immunoprecipitation
with antibodies directed against Tom20, Tom22, Tom40, and porin, or
antibodies derived from preimmune serum. The immunoprecipitates were
solubilized in sample buffer and analyzed as above.
In an initial cross-linking experiment, the homobifunctional reagent
DSG was added to OMV carrying pSu9-DHFR bound to either the
cis or the trans site. Several specific
cross-linking products were formed under both conditions (Fig.
1B). Some of the bands were obtained also by using the
"zero length" cross-linking reagent EDC (not shown; see below),
indicating that the preprotein is in intimate contact to these
cross-linking partners. The pattern of the cross-linking products in
the two binding conditions was similar, despite the fundamentally
different character of binding. Addition of chemical amounts of the
presequence peptide F1 (corresponding to amino acids
1-32 of the precursor of the
-subunit of yeast F1-ATPase) after the incubation of preprotein with OMV, but
before the cross-linking reaction largely reduced the formation of the specific cross-linking products in both the cis and
trans binding conditions (Fig. 1B), indicating
that the cross-links were occurring with a preprotein that is
specifically bound via its presequence.
The sizes of the cross-linking products suggested Tom20, Tom22, and Tom40 as possible partners. This expectation was confirmed by immunoprecipitation using antibodies directed against these TOM complex proteins (Fig. 1C). Two Tom40-specific cross-linking products were observed which differed in their electrophoretic mobility. These two cross-linking species correspond to different conformations of the preprotein-Tom40 complex.2 None of the cross-linking products was immunoprecipitated with antibodies derived from preimmune serum or with antibodies against porin, the most abundant protein of the mitochondrial outer membrane. We conclude from these results that a preprotein bound to the surface-exposed cis site is also in the vicinity of Tom40, a membrane-embedded component. Most likely, this early contact with Tom40 occurs through the presequence part of the preprotein (see also below). After transfer of the presequence into and across the outer membrane, the translocating polypeptide chain remains in the vicinity of both Tom40 and the receptors Tom20/Tom22.
To more precisely analyze the role of the presequence for preprotein
binding to the TOM machinery, we constructed two additional polypeptides. The first is a truncated version of pSu9-DHFR termed pSu9-DHFR13, consisting of the presequence of subunit 9 and
the N-terminal 13-amino acid residues of DHFR. The second, pSu9(+7),
consists of the presequence of subunit 9 plus 7 additional amino acid
residues resulting from the cloning procedure. Cross-linking of these
polypeptides to the TOM components by DSG or EDC can occur only via the
presequence part, as the short segments following the presequence lack
any lysyl residues. Addition of the cross-linkers to OMV that were incubated with pSu9(+7) under cis binding conditions
resulted in the formation of several specific cross-linking products
(Fig. 2A), while under conditions favoring
trans site binding, reduced amounts of pSu9(+7) were bound
to OMV, and only a few specific cross-linking bands were detected.
Similar results were obtained with pSu9-DHFR13 (data not
shown). As seen by immunoprecipitation, pSu9(+7) bound at the
cis site became cross-linked to Tom20, Tom22, Tom40, and Tom70 (Fig. 2B). Cross-linking to the latter two components
is consistent with results from a previous cross-linking study that utilized a presequence peptide to identify new components of the mitochondrial protein translocation machinery (25). The additional band
at an apparent molecular mass of 18 kDa (Fig. 2A)
corresponds most likely to the cross-linking product of the preprotein
with one of the small Tom proteins. Thus, a surface-bound presequence is in intimate contact with various TOM complex proteins including the
receptors. After the high salt wash only 13% of the preprotein remained bound to OMV as estimated by PhosphorImager analysis. The
cross-link to Tom40 relative to bound protein occurred to a similar
extent as under the cis binding condition (Fig.
2A). This result shows that a presequence polypeptide bound
in a salt-resistant fashion stays in the vicinity of Tom40. Further,
the data suggest that stable binding to the trans site
requires a contribution of the mature part of the preprotein.
We next investigated whether establishing a direct contact between the
presequence of a preprotein and Tom40 requires the participation of the
cytoplasmic domains of Tom20/Tom22 receptors. For that purpose, we
performed the cross-linking of pSu9-DHFR and pSu9(+7) with OMV that
were pretreated with trypsin. This pretreatment quantitatively removes
the surface receptors and completely abolishes cis site
binding (8), but leaves Tom40 intact.3
Binding to trypsin-treated OMV under the trans site
conditions was reduced to about 20-30% of the binding to untreated
OMV (Fig. 3, A and B). Using the
zero-length cross-linking reagent EDC, adducts between both preproteins
and Tom40 were observed with trypsin-pretreated OMV. As quantitated by
PhosphorImager analysis, the efficiency of cross-linking to Tom40
relative to bound precursor protein was about 2-fold higher than in the
presence of surface receptors. This suggests that a preprotein, via its
presequence, can bind to Tom40, even in the absence of surface
receptors. Thus, the surface receptors appear to be essential for
efficient accumulation of preproteins at the trans site (7),
but they are not necessary for establishing and maintaining the
interaction with Tom40.
We asked whether the interaction of the presequence with the different
TOM components is dependent on soluble factors that are contained in
the lysate mixture. Factors such as MSF and Hsp70 have been reported to
be important for establishing the association (reviewed in Ref. 26).
Chemical amounts of the purified presequence peptide F1
(see above) were bound to OMV. The OMV were washed with high or low
salt buffers and cross-linking with EDC was performed. Immunostaining
analysis revealed specific cross-linking of this presequence peptide to
Tom22 and to Tom40 (Fig. 4, A and
B, marked b). In addition, cross-linking of Tom22
and Tom40 to one of the small TOM complex proteins was seen (Fig. 4,
A and B, marked a). Weak cross-linking
of the presequence peptide was also observed to Tom20, but no products
were recognized for porin (not shown). Strikingly, the cross-link to
Tom22 was absent, when OMV were washed with high salt buffer, in
contrast to the cross-link to Tom40. It appears that F1
is bound to Tom22 and Tom40 in a distinct fashion, a situation
presumably reflecting the different binding properties of preproteins
such as pSu9-DHFR to the cis and trans sites (see
above). Cross-linking was specific for mitochondrial presequences, as
no reaction was observed for a control peptide (CH4),
corresponding to amino acids 1-25 of N. crassa cytochrome c heme lyase, which does not resemble a presequence (Fig.
4A) (cf. Ref. 27). The cross-linking product of
F1
with Tom40 was observed at a similar efficiency also
after trypsin pretreatment of the OMV (Fig. 4B),
corroborating our results from above that the presequence- Tom40
contact can occur independently of receptors. Taken together, our data
show that a presequence peptide bound to OMV is in intimate contact
with the surface receptors Tom20 and Tom22 only in the presence of low
salt, whereas the interaction with Tom40 may persist even after a high
salt wash. Since the results were obtained with a purified presequence
peptide, no soluble factors are necessary for establishing these
interactions.
It was now of particular interest whether the presequence-TOM protein
interactions observed with isolated OMV can be identified during the
import of preproteins into intact mitochondria. For that purpose,
import intermediates were accumulated with isolated mitochondria. To
avoid translocation across the inner membrane, both matrix ATP and the
membrane potential across the inner membrane were depleted (28,
29). pSu9-DHFR and pSu9(+7) were bound under conditions that result in
almost exclusive binding to the cis site and in binding to a
state corresponding to the trans site (7, 30,
31).2 As seen with isolated OMV, the DHFR moiety became
unfolded in the latter binding condition, a characteristic feature of
trans site binding.4 The
cross-linking pattern observed for binding of both preproteins under
cis site conditions was similar to that obtained with OMV, i.e. specific cross-links to Tom20, Tom22, and Tom40 were
formed (Fig. 5). After incubation of the preproteins
with mitochondria at 25 °C and a high salt wash (trans
condition), the cross-linking efficiency to the TOM components was
reduced, even though the amount of bound preproteins was comparable to
that under the cis site condition. This observation may be
due to the further movement of the presequence into the intermembrane
space or to the outer face of the inner membrane, a reaction that is
not possible in OMV. Nevertheless, preproteins bound under this
condition could be specifically cross-linked to Tom40 and to a lesser
extent to Tom20 and Tom22 (Fig. 5). Thus, the nearest neighbors of a
presequence during its transport across the outer membrane of isolated
mitochondria are the same as those identified with OMV. In summary, our
data suggest that the presequence of a translocating preprotein is in
the proximity of Tom40 under both the cis and
trans binding conditions. Since in our experiments, the zero
length cross-linker EDC was used, Tom40 appears to be in direct contact
with the presequence, especially when bound at the trans
site.
Our present cross-linking study analyzes the neighborhood of
presequence-containing preproteins bound to the protein import complex
of the mitochondrial outer membrane. The data allow us to extend and
refine our view of the molecular mechanism of preprotein translocation
across this membrane (see, e.g. Ref. 5). Earlier investigations have shown that the preprotein first interacts with the
surface receptors Tom20 and Tom22 (see Fig. 6,
stage A). This has been demonstrated by various techniques
including binding studies (8, 9) and cross-linking techniques (32). Our
study now shows that it is the presequence part of the preprotein which
is in close contact to the receptors. In this surface-bound stage
previously defined as the cis site, the presequence is also in contact with Tom40, a component, which according to its high protease resistance, is embedded in the outer membrane and most likely
forms the translocation channel or a major part thereof. This contact
could occur either via a surface-exposed region of Tom40 (Fig. 6,
stage A), or the presequence could be inserted at least
partially into the translocation pore thereby initiating the
translocation process (Fig. 6, stage B). Only N-terminal
parts of the presequence may have entered the translocation channel in
this surface-bound state, since the mature part of the preprotein remains fully folded, and added matrix processing peptidase can readily
cleave off the presequence of cis site-bound preproteins (7)
(see Fig. 6, stages A and B).
Deeper insertion of the presequence across the outer membrane requires the unfolding of the mature part of the preprotein, a reaction which does not depend on external energy sources such as ATP hydrolysis (Fig. 6, stage C) (7). Translocation is thought to be driven by the interaction of the presequence with a second presequence-specific binding site. This site was previously termed trans site, since the presequence is accessible from the intermembrane space side of the outer membrane. The matrix processing peptidase introduced into the lumen of OMV could cleave off the presequence of trans site-bound preproteins, but was unable to do so from the cytosolic side (Fig. 6, stages C and D) (7). Our cross-linking study now provides important information about the molecular environment forming this presequence-specific binding site. Tom40 appears to be the major partner of interaction for the presequence at this stage. The presequence-Tom40 interaction can be seen even after removal of the cytoplasmic domains of the receptor proteins, suggesting that these domains are not essential for the formation of this binding site. Moreover, cross-linking of a presequence peptide to Tom40 was observed also after removing loosely bound molecules by a high salt wash (Figs. 2 and 4). In contrast, cross-linking to Tom22 was almost completely abolished under such conditions. Thus, Tom40 appears to be directly involved in forming the second presequence binding site. Preproteins bound to this stage remain in close contact to Tom20 and Tom22. This contact is formed mainly through the mature part of the preprotein, as cross-linking of trans site-bound preproteins to Tom20 and Tom22 is only seen with preproteins containing a larger portion of the mature part. In addition to Tom40, other components of the TOM complex may be important for the formation of the trans presequence binding site. One such component is likely one of the small TOM complex proteins that was observed to become cross-linked to a presequence polypeptide.
The precise position of the trans presequence binding site on Tom40 remains unknown. Based on the accessibility of the matrix processing peptidase cleavage site of trans site-bound preproteins from the inner face of the outer membrane, it may be argued that the trans site is located toward the intermembrane space. However, a situation in which the presequence is in dynamic equilibrium between the translocation pore and the intermembrane space side seems also possible (Fig. 6, stages C and D). A prerequisite for defining the binding site in detail is the unraveling of the structure of Tom40.
Earlier investigations have addressed the role of the intermembrane space domain of Tom22 in presequence binding on the trans side of the outer membrane. Two studies found no significant alterations in preprotein translocation across the outer membrane upon deletion of this segment of Tom22 (33, 34). In another report, deletion of this piece had a dramatic influence on the growth of mutated yeast cells and largely affected protein import into mitochondria (10). In addition, an interaction between presequences and the C-terminal portion of yeast Tom22 was found. In contrast, no significant association between presequences or preproteins and the corresponding piece of N. crassa Tom22 was observed (34). Our present cross-linking study was unable to detect any cross-linking of Tom22 to presequence peptides, when they were bound at the salt-resistant trans site of the outer membrane. Thus, our previous (34) and present results suggest that the C terminus of Tom22 does not play an essential role in binding and translocation of mitochondrial presequences across the mitochrondrial outer membrane.
Preprotein translocation across the outer membrane of mitochondria is similar in a number of aspects to protein transport across the chloroplast outer envelope membrane (35). The N-terminal targeting information is first recognized at the organellar surface. In chloroplasts, the outer envelope protein IAP86/OEP86 serves as the initial binding partner (36, 37). As in the case of the mitochondrial outer membrane, the surface-bound preprotein is in close proximity to a membrane-embedded component that possibly forms a translocation pore, namely IAP75/OEP75 (37, 38). Insertion of the targeting sequence leads to a stable import intermediate in which the presequence is recognized a second time. In both organelles, the membrane-embedded pore-forming components (Tom40 in mitochondria or IAP75/OEP75 in chloroplasts) are involved in the formation of these two binding sites.
What may be the relevance of a second presequence binding site for the protein import reaction? Repeated deciphering of the targeting information might be important to increase the accuracy of targeting. At the organellar surface, preproteins are bound in a rather labile fashion and readily equilibrate with the bulk solution. The labile character of interaction in this first binding site would be well suited for selecting the cognate preproteins from the large pool of cytosolic proteins. Erroneously bound proteins could readily dissociate from the organellar surface at this stage. Efficient insertion into the translocation pore and binding to the second presequence binding site is only feasible if the correct targeting information is attached to the N terminus of the preprotein. It is possible that different features of the targeting sequences are recognized at these two binding sites. Such a "double-check system" would thus guarantee the accurate selection of the correct preproteins.
We thank Dr. B. Westermann for providing the plasmid encoding the pSu9(+7) protein and P. Heckmeyer and M. Braun for excellent technical assistance.