* Abteilung Biochemie II, Zentrum Biochemie und Molekulare Zellbiologie, Georg-August-Universität, 37073 Göttingen,
Germany; and Harvard Medical School, Department of Cell Biology, Boston, Massachusetts 02115
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Abstract |
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The Sec61 complex is the central component
of the protein translocation apparatus of the ER membrane. We have addressed the role of the subunit
(Sec61
) during cotranslational protein translocation.
With a reconstituted system, we show that a Sec61 complex lacking Sec61
is essentially inactive when elongation and membrane targeting of a nascent chain occur
at the same time. The translocation process is perturbed
at a step where the nascent chain would be inserted into
the translocation channel. However, if sufficient time is
given for the interaction of the nascent polypeptide
with the mutant Sec61 complex, translocation is almost normal. Thus Sec61
kinetically facilitates cotranslational translocation, but is not essential for it.
Using chemical cross-linking we show that Sec61
not only interacts with subunits of the Sec61 complex
but also with the 25-kD subunit of the signal peptidase
complex (SPC25), thus demonstrating for the first time
a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61
and SPC25 and between Sec61
and Sec61
depend on the
presence of membrane-bound ribosomes, suggesting
that these interactions are induced when translocation
is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.
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Introduction |
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IN higher eukaryotes, most proteins are transported
across the ER membrane in a cotranslational manner.
The translating ribosome binds tightly to the ER
membrane (Connolly et al., 1989; Crowley et al., 1993
,
1994
; Kalies et al., 1994
), and the growing nascent polypeptide chain is transferred directly from the channel in the ribosome into a channel in the membrane (Blobel and
Dobberstein, 1975
; Connolly et al., 1989
; Simon and Blobel, 1991
; Görlich et al., 1992b
; Görlich and Rapoport,
1993
).
The cotranslational translocation pathway in mammals
can be reproduced with reconstituted proteoliposomes
containing only three membrane protein components: the
SRP receptor complex, the translocating chain-associating
membrane (TRAM)1 protein, and the Sec61 complex
(Görlich and Rapoport, 1993). The signal recognition particle (SRP) receptor is required to target a ribosome-
nascent chain complex to the ER membrane. The function
of the TRAM protein is still unclear; it is only required for
the translocation of a subset of proteins (Görlich et al., 1992a
; Voigt et al., 1996
). The Sec61 complex represents
the essential core of the translocation machinery in the ER
membrane. It consists of three subunits, an
subunit
(Sec61
) that spans the membrane 10 times, and
and
subunits (Sec61
and Sec61
, respectively) that each span
the membrane a single time.
The Sec61 complex is evolutionarily highly conserved
and is proposed to carry out at least three different functions. First, it is the major constituent of the protein conducting membrane channel. Cross-linking experiments have
shown that its subunit is in continuous proximity of nascent polypeptide chains passing through the membrane
(Mothes et al., 1994
). Recent electron microscopic data
demonstrate that the Sec61 complex can form cylindrical
oligomers that presumably represent the channels (Hanein et al., 1996
; Beckmann et al., 1997
). Second, the Sec61
complex is tightly associated with membrane-bound ribosomes and is likely to be the ribosome receptor (Görlich
et al., 1992b
; Kalies et al., 1994
; Jungnickel and Rapoport,
1995
). Third, the Sec61 complex is involved in a signal sequence recognition event that takes place inside the membrane (Jungnickel and Rapoport, 1995
).
The role of the Sec61 complex subunits for its different
functions is not yet clear. Whereas the presence of a multi-spanning subunit in a channel-forming protein complex
may not be surprising, the role of the two small single-spanning polypeptide chains remains mysterious. A particular enigma is the
subunit. In Saccharomyces cerevisiae,
the simultaneous deletion of the
subunits of the two homologous Sec61 complexes (Sec61p and the Ssh1p complex) is not lethal; the cells only show a growth defect at elevated temperatures (Finke et al., 1996
). Thus, despite the
fact that the protein is evolutionarily highly conserved, it
does not appear to be absolutely required in vivo. Posttranslational protein transport across yeast ER membranes in vitro is reduced, but not completely prevented,
when the
subunits are lacking. At which point the transport process is inhibited, and whether the
subunits play any role in the cotranslational mode of protein transport
has not yet been investigated.
The function of the subunit of the Sec61 complex may
not necessarily be restricted to the actual translocation
process. The transport of proteins across the ER membrane must be intimately coupled to their modification
and folding, and it seems possible that the
subunit could
be involved in interactions between the translocation
channel and modifying enzymes or chaperones. A physical association of these proteins with the channel may enhance the efficiency of their function. A good example is
the signal peptidase, an abundant enzyme whose active
site is in the lumen of the ER. Although it can cleave the
signal peptide of even completed polypeptide chains, its
efficiency is probably much higher when it can act on
polypeptide chains that are just emerging from the translocation channel into the lumen of the ER. One may therefore predict its physical association with channel constituents. The signal peptidase complex is composed of five
membrane protein subunits (Evans et al., 1986
), two with
an active site for enzymatic activity, one with a lumenal
domain of unknown function, and two (12 and 25 kD) with
cytosolic domains. The function of the latter is particularly unclear because they cannot contribute to the enzymatic
activity on the lumenal side of the membrane, and are not
essential for the viability of yeast cells (Lively and Walsh,
1983
; Fang et al., 1996
; Kalies and Hartmann, 1996
; Mullins et al., 1996
). They would be especially good candidates
to serve as linkers to the translocation channel.
In the present paper, we have analyzed the role of the subunit of the Sec61 complex in the cotranslational translocation pathway in mammals. Using a reconstituted system, we demonstrate that the
subunit kinetically facilitates, but is not essential for, cotranslational translocation.
The protein does not play a role in the interaction of the
ribosome with the Sec61 complex, but rather in the insertion of the nascent chain into the translocation site. Using
a bifunctional cross-linker, we also provide evidence that it
interacts specifically with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the
first time a tight interactions between the SPC and the
Sec61 complex. Cross-linking is only observed in the presence of membrane-bound ribosomes. These data thus suggest that, upon ribosome binding to the Sec61 complex, an
interaction between the cytosolic domains of the Sec61
and SPC25 serves to recruit the signal peptidase complex
to the translocation site. Together with the observation
that cross-linking between the
and
subunits of the
Sec61 complex is also dependent on translocation, these
data provide first evidence for structural changes in the
translocation apparatus upon initiation of cotranslational translocation.
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Materials and Methods |
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Preparation of Reconstituted Proteoliposomes
Microsomes treated with puromycin and high salt (PK-RM) (Görlich and
Rapoport, 1993) were solubilized at a concentration of 2 eq/µl in SB (50 mM
Hepes/KOH, pH 7.6, 15% glycerin, 400 mM potassium acetate, 10 mM
magnesium acetate, and 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 5 µg/ml
chymostatin as protease inhibitors) containing 1.5% decylsucrose for 30 min on ice. After centrifugation for 10 min at 14,000 rpm in a microfuge,
0.7% deoxyBigChap was added to the supernatant. 500 µl detergent extract was mixed with 100 µl of an immunoaffinity resin that contained 0.2 mg/ml affinity-purified antibodies directed against the NH2 terminus of
Sec61
, covalently coupled to protein A-Sepharose (Görlich and Rapoport, 1993
). The column was previously equilibrated with SB containing
1.5% decylsucrose and 0.7% deoxyBigChap. The incubation was done for 18 h in an overhead shaker in a cold room. Every 6 h the resin was replaced with a new one. The non-bound material was collected and proteoliposomes were produced by incubation with SM2-biobeads at 4°C (Bio
Rad Laboratories, Hercules, CA) (Görlich and Rapoport, 1993
). Proteoliposomes containing the purified Sec61 complex were prepared as described previously (Kalies et al., 1994
).
The signal peptidase complex (SPC) was purified as reported (Görlich
and Rapoport, 1993). Proteoliposomes were produced by mixing 20 µl of
the SPC preparation (200 eq) with 20 µl of a phospholipid mixture (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol in a ratio of 100:25:3:12.5; total concentration 5 mg/ml). 40 µg
SM2 biobeads equilibrated in SB were added and vesicles were prepared
as described (Görlich and Rapoport, 1993
).
Binding of Ribosomes to Proteoliposomes
The purification, radioactive labeling, and binding of ribosomes to reconstituted proteoliposomes were done as described (Kalies et al., 1994). The
ribosome binding was carried out at 26°C.
Cross-linking with Bis-maleimidohexane
A 25 mM stock solution of bis-maleimidohexane (BMH) in dimethylformamid was prepared. Microsomes or proteoliposomes were incubated with
increasing amounts of BMH for 30 min at 0°C in a cross-link buffer containing 50 mM Hepes/KOH, pH 7.6, 100 mM KCl, 5 mM MgCl2, 250 mM
sucrose, and protease inhibitors. The reaction was quenched by addition
of 100 mM -mercaptoethanol and the samples were analyzed by SDS-PAGE and immunoblotting, using various antibodies.
Transcription, Translation, and Translocation
Transcripts coding for preprolactin were produced by in vitro transcription with T7 RNA polymerase of the plasmid pGEMBP1. For full-length transcripts, the plasmid was cut with PstI and in the case of the transcripts coding for the 86-mer with PvuII.
Translation of the full-length protein was carried out in the wheat germ system in the presence of 40 nM SRP, [35S]methionine, and membranes at 26°C for 30 min. After 15 min, 4 µM edeine was added to prevent further initiation. In a parallel experiment translation was started in the absence of membranes for 15 min at 26°C. 4 µM edeine and membranes were then added. The samples were incubated on ice for 10 min followed by an incubation at 26°C for additional 15 min. Half of the sample was precipitated with 15% TCA. The other half was treated with 0.5 mg/ml proteinase K for 30 min on ice, before precipitation with TCA. The pellets were washed with acetone and dissolved in SDS sample buffer. After SDS-PAGE, the gels were dried and analyzed in a quantitative manner with a PhosphorImager (Bas 1000; Fuji, Tokyo, Japan).
Translation of the 86-mer of preprolactin was done in the reticulocyte lysate system in the presence of [35S]methionine at 24°C for 20 min. For the mock translation, the mRNA was omitted. 2 mM cycloheximide and 5 eq membranes were added to 300 µl translation mix and the incubation was continued at 0°C for 10 min and at 25°C for 5 min. After centrifugation in a microfuge at 14,000 rpm for 5 min the samples were layered on top of a sucrose cushion (400 µl of 50 mM Hepes/KOH, pH 7.6, 100 mM KCl, 5 mM MgCl2, and 500 mM sucrose) and were centrifuged at 75,000 rpm for 20 min (rotor TLA 100.3; Beckman Instruments, Inc., Fullerton, CA). Finally the pellets were resuspended in 65 µl cross-link buffer.
Immunoblotting, Immunoprecipitation, and Antibodies
Immunoblotting was carried out as described (Görlich et al., 1992b). Before immunoprecipitation, the samples were denatured in SDS sample
buffer omitting reducing reagents. The samples were then mixed with 10 vol of SB containing 1% Triton X-100, and incubated for 20 min on ice.
Affinity-purified antibodies against Sec61
coupled to protein A-Sepharose
were added. After shaking in a cold room for 3 h, the antibody resin was
washed with 1% Triton X-100 in SB. Finally, the material bound to the
column was analyzed by SDS-PAGE and Western blotting.
Polyclonal antibodies directed against the following synthetic peptides
were used: against the NH2 terminus of Sec61 (Görlich et al., 1992b
);
against the COOH terminus of Sec61
(Görlich and Rapoport, 1993
);
against the position 137 to 150 of the SRP-receptor
(docking protein
);
against the COOH terminus of the TRAM protein (Görlich et al., 1992a
);
against the NH2 terminus of Sec61
(DQVMQFVEPSRQC); and against
the COOH terminus of SPC25 (Kalies and Hartmann, 1996
).
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Results |
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Sec61 Facilitates Cotranslational
Protein Translocation
We used reconstituted proteoliposomes, immunodepleted
of the subunit of the Sec61 complex, to investigate the
role of the protein in cotranslational protein translocation.
Mammalian PK-RM, were solubilized in a detergent mixture that leads to the dissociation of the Sec61 complex
into its subunits. The detergent extract was incubated either with antibodies directed against Sec61
that had been
immobilized on protein A-Sepharose or, to generate a mock-depleted extract, with protein A-Sepharose alone.
The efficiency of immunodepletion was tested by Western
blotting, using a radioactively labeled secondary antibody
and a PhosphorImager. In a typical experiment, the depleted
proteoliposomes contained <0.5% of the original amount
of Sec61
, whereas all other proteins tested remained almost unaffected (Fig. 1 A). The only exception was
Sec61
, the concentration of which was reduced to 50-
70% in the worst case. Apparently, under the conditions
used, the
and the
subunits are not totally dissociated.
|
We then tested the reconstituted proteoliposomes for
their ability to translocate proteins synthesized in a wheat
germ translation system. Microsomes (PK-RM) served as
a control. Transcripts coding for full-length preprolactin
were translated at 26°C in the presence of microsomes or
proteoliposomes, the concentration of which was normalized for their Sec61 content (Fig. 1 A). PK-RM and
mock-depleted proteoliposomes had the same translocation activity (Fig. 1 B, lanes 6 and 7), whereas the depleted
proteoliposomes were totally inactive (lane 8). In the absence of Sec61
, processed prolactin was produced (Fig. 1
B, lane 4) that, however, was accessible to the action of
proteinase K; it therefore presumably represents material
generated by signal peptidase that was incorporated into the reconstituted membrane in the inverse orientation.
Since Sec61 is not essential in yeast, we wondered if
the mammalian Sec61 complex lacking this component
may show in vitro translocation activity under less stringent conditions than used before. We therefore performed
the translocation reaction such that more time would be
allowed for the membrane binding of the ribosome-
nascent chain complex. Translation of the full-length transcript coding for preprolactin was initially carried out in the presence of SRP but absence of membranes. This leads
to a translational arrest when the polypeptide chain
reaches a length of ~70 residues. The membranes were
then added at 0°C, conditions that allow efficient membrane binding of the ribosome-nascent chain complexes
but no chain elongation. The samples were then warmed
up to 26°C to continue translation and concomitant translocation. With this protocol, proteoliposomes lacking Sec61
were active in translocation, although their activity was
lower by a factor of three compared with the wild-type
complex (Fig. 1 B, lane 16 vs. lane 15). Thus, the depleted
proteoliposomes are capable to translocate polypeptides if
given enough time in the membrane targeting reaction.
The results also indicate that the Sec61 complex lacking its
subunit has not been irreversibly denatured during the
prolonged immunodepletion procedure. These data suggest that the ribosome-nascent chain complex was targeted to the membrane and thus brought in contact with
the signal peptidase, but that a subsequent translocation
step was perturbed. We also found that a fragment of preprolactin of 86 amino acids could be efficiently targeted to
reconstituted proteoliposomes and reached a protease-protected state even if Sec61
was lacking (data not
shown), supporting the conclusion that insertion of the nascent chain into the Sec61
-depleted translocation site can
occur if no chain elongation is going on.
It should be noted that in the absence of membranes, almost no full-length preprolactin could be observed (Fig. 1
B, lanes 1 and 9), indicating that under the conditions
used, SRP produced a tight translational arrest. Both the
microsomes and the two types of proteoliposomes were
able to release the translational arrest (Fig. 1 B, lanes 2-4
and 10-12), indicating that this reaction is not dependent
on the presence of Sec61.
Sec61 Is Not Required for Ribosome Binding
Our data suggested that in the absence of Sec61, the
binding of the ribosome-nascent chain complex to the ER
membrane is less efficient. This could be due to either a
defect in the interaction of the mutated Sec61 complex
with the ribosome, or to a perturbed insertion of the nascent chain into the translocation site. We therefore analyzed whether the
subunit plays a role in ribosome binding. Depleted and mock-depleted proteoliposomes were
incubated at physiological salt concentrations with radioactively labeled ribosomes and increasing amounts of unlabeled ribosomes, both lacking nascent polypeptide
chains. Under these conditions, the ribosomes interact
mainly with the Sec61 complex (Kalies et al., 1994
). To
separate the unbound from the bound fraction, the membranes were floated in a sucrose gradient. Scatchard plot
analysis was used to estimate the number of binding sites
and the apparent dissociation constants (Fig. 2). Both the
depleted and mock-depleted proteoliposomes were found
to bind ribosomes with approximately the same binding
constant. Also, the number of binding sites was about the
same. The measured parameters are in good agreement with published data for the binding of ribosomes to PK-RM
and proteoliposomes (Kalies et al., 1994
), although the dissociation constants seem to be somewhat higher at 26°
than at 0°C. Thus, steps other than the interaction between
SRP and its membrane receptor or the binding of ribosomes to the Sec61 complex must be responsible for the
less efficient insertion of the nascent polypeptide chains
into proteoliposomes lacking Sec61
.
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Sec61 Interacts with the 25-kD Subunit of the Signal
Peptidase Complex
To further analyze the function of Sec61, we investigated
its molecular environment in the membrane by chemical
cross-linking. Rough microsomes (RM) were treated with
increasing amounts of BMH, a bifunctional cross-linking
reagent that reacts with sulfhydryl (SH) groups. The proteins were subsequently separated by SDS-PAGE and analyzed by immunoblotting with antibodies against Sec61
(Fig. 3, lanes 2-6). Three cross-linked products were detected with the antibodies (Fig. 3, lanes 2-6 vs. lane 1). The
apparent molecular weights of the cross-linked proteins
were estimated to be 12, 23, and 38 kD, assuming an apparent molecular weight for Sec61
of 13 kD.
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To identify the cross-linked proteins, microsomes were
treated with two different concentrations of BMH and dissolved in SDS-containing buffer to dissociate noncovalent
chemical bonds. The extract was subjected to immunoprecipitation with Sec61 antibodies and the precipitated material was analyzed by Western blotting using different antibodies. Fig. 3 shows the immunoblots with antibodies
directed against Sec61
(Fig. 3, lanes 7-10), Sec61
(Fig. 3,
lanes 11-14) and SPC25 (lanes 15-18). The product containing the 38-kD protein could be immunoprecipitated
with Sec61
antibodies (Fig. 3, lane 10) and was recognized by the Sec61
antibody (lane 14), indicating that it is
generated by cross-linking between the
and
subunits of the Sec61 complex. The product containing a protein of
~23 kD could be immunoprecipitated with Sec61
antibodies (Fig. 3, lane 9) and reacted with antibodies against
SPC25 (lane 17) and is thus generated by cross-linking between these two proteins. Neither Sec61
nor SPC25 were
coprecipitated with Sec61
if BMH was omitted (Fig. 3,
lanes 12 and 16), and both antibodies recognized single
bands in untreated RM (lanes 11 and 15).
The product containing the protein of ~12 kD did not
react with any of the antibodies tested. Considering its
size, we suspected that it may represent a product generated by cross-linking of two subunits of the Sec61 complex. To test this assumption, purified Sec61 complex was
reconstituted into proteoliposomes and subjected to cross-linking with BMH. When analyzed by SDS-PAGE and immunoblotting with Sec61
antibodies, a cross-linked product containing a 12-kD protein was again observed (Fig. 4
A, lanes 10-12), indicating that the cross-linking partner is
indeed a constituent of the Sec61 complex. The smaller
cross-linked product in Fig. 4 A, lanes 11 and 12 (marked
with an asterisk) is probably generated by cross-linking between Sec61
and Sec61
, the smallest subunit of the
Sec61 complex. The appearance of this cross-linked product in native microsomes was variable among different experiments.
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Ribosome-dependent Structural Changes of the Translocation Site
We were concerned that the membrane-bound ribosomes
may prevent full access of the bifunctional cross-linker to
Sec61 so that only a subset of its interacting partners
could be detected. However, to our surprise, when PK-RM
were used in cross-linking experiments, not only were no
additional cross-links observed, but those between Sec61
and Sec61
and between Sec61
and SPC25 could no
longer be seen (Fig. 4 A, lanes 4-6). Treatment of RM with
high salt or puromycin alone did not change these cross-links (data not shown), suggesting that their disappearance
requires the dissociation of the ribosomes into subunits. In
agreement with this assumption, proteoliposomes reconstituted from a crude detergent extract of microsomes or
proteoliposomes containing only the purified Sec61 complex, which both lack membrane-bound ribosomes, also
did not give these cross-links (Fig. 4 A, lanes 7-12).
A similar conclusion could be drawn when the cross-linking reaction was analyzed with SPC25 antibodies (Fig.
4 B). With RM two cross-linked products were seen, one
of ~36 kD between SPC25 and Sec61, and another of
~46 kD (Fig. 4 B, lane 2 vs. lane 1). With PK-RM or with
proteoliposomes reconstituted from a crude detergent extract, the adduct of SPC25 and Sec61
was no longer observed (Fig. 4 B, lanes 4-6 and lanes 7-9, respectively),
whereas the 46-kD cross-linked product remained unchanged. The latter was also observed with proteoliposomes containing only the purified signal peptidase complex
(Fig. 4 B, lane 11). Two SPC subunits, the nonglycosylated
SPC25 and the glycoprotein SPC22/23 carry SH groups
and have an appropriate molecular weight to produce this
46-kD cross-link with SPC25. As the molecular weight of
the cross-linked product did not change after treatment
with N-glycosidase F (data not shown), we conclude that it
consists of two SPC25.
To exclude that the high salt treatment during the preparation of PK-RM was responsible for the structural alterations identified, ribosomes were detached from the membrane by an independent method. When the reaction with
BMH was performed in the presence of 10 mM EDTA under low salt conditions, the cross-link between Sec61 and
Sec61
and that between Sec61
and SPC25 could not be
seen anymore (Fig. 5, lanes 5 and 4 vs. lanes 2 and 3).
However, the homotypic cross-link between two Sec61
remained unchanged, indicating that the EDTA did not
interfere with the reactivity of the BMH. It should be
noted that the extent to which the cross-linking intensity
was reduced varied in different experiments.
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If the ribosome-dependent alteration of the cross-link
pattern has a real physiological significance it should be
possible to reproduce the cross-link between Sec61 and
Sec61
and that between Sec61
and SPC25 by a retargeting of ribosomes carrying nascent polypeptide chains at ribosome free membranes. Ribosome-nascent chain complexes were produced by an in vitro translation of truncated mRNA coding for the first 86 amino acids of preprolactin
(86-mer). Ribosome-free membranes (PK-RM) were then
added to the translation mix (Fig. 6 A, lanes 4-6). As controls PK-RM and RM were incubated with a translation
mix that did not contain any preprolactin mRNA (Fig. 6
A, lanes 1-3 and lanes 7-9). After isolation of the membranes aliquots of each sample were treated with different
amounts of BMH. The samples were analyzed by Western
blotting with Sec61
antibodies using enhanced chemiluminescence as a detection system (Fig. 6 A) or by quantitative immunoblotting using radioactively labeled secondary
antibodies and a PhosphorImager (Fig. 6 B). The quantitation (Fig. 6 B) shows that the incubation of PK-RM with
ribosome-nascent chain complexes led to a clear restimulation of the cross-link intensity between Sec61
and Sec61
and between Sec61
and SPC25. Similar results were obtained if EDTA-treated membranes were analyzed (data
not shown).
|
Taken together, these data provide evidence that the subunit of the Sec61 complex is involved in ribosome-
dependent conformational changes of the translocation
channel and that it specifically interacts with the signal
peptidase during cotranslational translocation.
![]() |
Discussion |
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In this paper, we have studied the role of the subunit of
the Sec61 complex during cotranslational protein translocation into the mammalian ER. We have found that the
translocation competence of reconstituted proteoliposomes
immunodepleted of Sec61
is greatly reduced when tested
under conditions in which membrane targeting and translation occur at the same time. However, if ribosomes carrying short nascent chains are first targeted to the membrane before translation is continued, i.e., if enough time is
given for their membrane insertion, the depleted proteoliposomes are only marginally reduced in their activity.
These results can be explained by the assumption that
elongation of a nascent chain and its insertion into the
translocation channel are kinetically competing processes:
if the membrane insertion is too slow, elongation of the
nascent chain would continue in the cytosol and its folding would prevent a later interaction of the signal sequence
with the translocation apparatus. We therefore infer that
the
subunit is required for a rapid insertion of the ribosome-bound nascent chain into the translocation sites in
the ER membrane. The observation that the
subunit is
not essential is in agreement with the fact that the deletion
of the two
subunits in yeast cells is not lethal and leads to
a growth phenotype only at elevated temperatures. Since
microsomes isolated from the mutant had a reduced activity for posttranslational protein transport, these and the
present results indicate that the
subunit plays a role in
both pathways, but is not absolutely required in either one.
As the cotranslational translocation process is known in
much detail, we have been able to analyze the effect of
Sec61 depletion on the various steps. One of the first
steps is the interaction of the ribosome-nascent chain-
SRP complex with the SRP receptor in the membrane that
leads to the release of the translational arrest exerted by
SRP. This reaction was not perturbed by the depletion of
Sec61
, although previous data showed that, for optimal release, both the SRP receptor and the Sec61 complex are
required (Görlich and Rapoport, 1993
). We therefore conclude that the
subunit is not essential for this activity of
the Sec61 complex. The binding of ribosomes to the ER
membrane was also not affected by the absence of Sec61
.
Both the number of binding sites and the dissociation constant remained unchanged and were almost identical to
the values determined for the reconstituted, purified wild-type Sec61 complex. These results are consistent with the
observation that proteolytic degradation of the cytosolic
domain of Sec61
in microsomes does not prevent the
binding of ribosomes (Kalies et al., 1994
). Taken together,
our results indicate that even in the absence of Sec61
, efficient release of the SRP arrest and binding of the ribosome-nascent chain complex to the Sec61 complex can
occur. This conclusion is further supported by the observation that the membrane targeted nascent chains undergo signal peptide cleavage by signal peptidase located in the
wrong orientation in the reconstituted membrane. It
therefore appears that a step subsequent to membrane targeting, most likely the step in which the nascent chain is inserted into the Sec61 channel, is perturbed in the absence
of the
subunit. Structural changes in the Sec61 channel,
which may be required for its opening (Crowley et al.,
1994
; Jungnickel and Rapoport, 1995
), may occur with a reduced rate.
To further analyze the function of Sec61, we have
probed its molecular environment in ER membranes by
cross-linking. A bifunctional cross-linker was used that reacts specifically with SH groups and therefore can be expected to give a relative simple cross-linking pattern. As
predicted, in rough microsomes Sec61
was found in close
proximity to the
subunit of the Sec61 complex. We also found a cross-linked product consisting of two Sec61
molecules, perhaps explained by the occurrence of oligomers of the trimeric Sec61 complex in microsomes (Hanein et al., 1996
). Most interestingly, however, a specific
cross-linked product containing SPC25 was observed.
Conversely, when the cross-linking partners of SPC25 were analyzed, the only partner outside the signal peptidase complex was found to be Sec61
. Remarkably,
70% of SPC25 could be cross-linked to Sec61
. We
therefore believe that the cross-link between the two proteins indicates their specific interaction. These data provide the first evidence that the signal peptidase physically contacts the protein conducting channel in the membrane.
The cross-linking between Sec61
and SPC25 has likely
occurred between their cytosolic domains since the only
cysteine in Sec61
is in its NH2 terminus and two appropriately located cysteines exist in the NH2-terminal domain of SPC25 (Fig. 7), previously shown to be cytosolic
(Kalies and Hartmann, 1996
). SPC25 has almost no amino
acid residues in the lumen of the ER where the active site
of the signal peptidase resides (Fig. 7) and its function was
therefore obscure. On the basis of our data, we propose
that it is involved in an interaction with the Sec61 complex
to bring the enzyme close to the translocating polypeptide
substrate. However, sequestration in the translocation site
may not be absolutely essential for its function because the
signal peptidase complex is very abundant and can probably reach its substrates by mere diffusion in the plane of
the membrane, explaining why neither SPC25 nor Sec61
are essential for the viability of yeast cells.
|
Further evidence for our conjecture that Sec61 may be
involved in recruiting the signal peptidase complex to the
translocation site comes from cross-linking experiments in
which ribosome-stripped membranes were used. These
membranes gave many cross-link-reduced cross-links
between Sec61
and SPC25. After retargeting of ribosome-nascent chain complexes at almost ribosome-free
membranes, the cross-linking between both proteins was
increased, suggesting that their interaction is induced
when translocation is initiated by ribosome binding. The
cross-linking between Sec61
and Sec61
was also dependent on the presence of membrane-bound ribosomes.
Both effects required the dissociation of the ribosome, and
not merely the release of the nascent chain from the ribosome, since they occurred with puromycin at high but not
at low salt concentrations. The changes in cross-linking
pattern upon ribosome removal or retargeting of ribosome-nascent chain complexes could be caused by conformational alterations in an assembled complex or by dissociation of an assembly into subcomplexes. In any case,
they provide first evidence for structural changes among
known components of the translocation apparatus induced
by the onset of cotranslational translocation.
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Footnotes |
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Received for publication 11 April 1997 and in revised form 10 February 1998.
The work was supported by a grant from the Deutsche Forschungsgemeinschaft.We thank A. Wittstruck and B. Nentwig for technical help.
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Abbreviations used in this paper |
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BMH, bis-maleimidohexane; PK-RM, puromycin and high salt-treated rough microsomes; RM, rough microsomes; SPC, signal peptidase complex; SPC25, the 25-kD subunit of the signal peptidase complex; SRP, signal recognition particle; TRAM, translocating chain-associating membrane.
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References |
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