From the School of Biological Sciences, University of Manchester,
2.205 Stopford Building, Oxford Road, Manchester M13 9PT, United
Kingdom, and Medizinische Biochemie und
Molekularbiologie, Universität des Saarlandes,
Homburg D-66421, Germany
Received for publication, September 30, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Tail-anchored proteins are a distinct
class of membrane proteins that are characterized by a C-terminal
membrane insertion sequence and a capacity for post-translational
integration. Although it is now clear that tail-anchored proteins are
inserted into the membrane at the endoplasmic reticulum (ER), the
molecular basis for their integration is poorly understood. We have
used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find
that several newly synthesized tail-anchored proteins are transiently
associated with a defined subset of cellular components. Among these,
we identify several ER proteins, including subunits of the Sec61
translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal
peptidase complex. When we analyze the cotranslational membrane
insertion of a comparable signal-anchored protein we find the nascent
polypeptide associated with a similar set of ER components. We conclude
that the pathways for the integration of tail-anchored and
signal-anchored membrane proteins at the ER exhibit a substantial
degree of overlap, and we propose that this reflects similarities
between co- and post-translational membrane insertion.
Membrane protein insertion at the mammalian ER occurs most
commonly via the cotranslational pathway, in which a hydrophobic signal
sequence emerges from the ribosome and is recognized by the signal
recognition particle (SRP)1
(1). The ribosome-nascent chain-SRP complex is then targeted to the ER
membrane via an association with a cognate receptor complex (1, 2).
Upon its arrival at the ER, the nascent membrane protein is delivered
to the Sec61 translocon. This comprises multiple Sec61 heterotrimers,
composed of In contrast to higher eukaryotes, SRP-independent, post-translational
translocation plays a significant role in the yeast Saccharomyces
cerevisiae (7, 8). In this instance the precursors use cytosolic
chaperones to maintain translocation competence (9), and signal
sequence recognition occurs at the Sec complex of the ER membrane (10,
11). This Sec complex is made up of the heterotrimeric Sec61 complex
together with four other membrane-associated components, namely Sec62p,
Sec63p, Sec71p and Sec72p, as well as the ER luminal chaperone Kar2p
(the S. cerevisiae equivalent of BiP) (12-14). It has
become apparent that Sec62p and Sec63p are not restricted to S. cerevisiae, and similar proteins have been identified in mammals,
although their precise function is unknown (15, 16). Specific examples
of post-translational translocation have been identified in higher
eukaryotes, although these tend to be the exception rather than the
rule. In the case of very short presecretory proteins, such as
prepromelittin, the N-terminal signal sequence does not have an
opportunity to interact with SRP before translation is terminated (17,
18). Hence, prepromelittin translocation is independent of SRP but
dependent upon cytosolic component(s) and ATP, presumably to maintain
the polypeptide in a "translocation-competent" state (18).
Tail-anchored proteins form a distinct class of integral
membrane proteins, possessing a single membrane insertion sequence at
their C terminus and displaying their remaining N-terminal portion in
the cytosol. The majority of tail-anchored proteins become integrated
at the ER membrane (19-21, but see also Ref. 22), and members of this
class carry out a range of important cellular functions, such as ER
translocation (Sec61 The membrane insertion sequence of tail-anchored proteins acts as an ER
targeting signal, and its relative position dictates that membrane
insertion is post-translational and hence likely to be SRP-independent
(9, 26). To date, only general characteristics regarding the targeting
and membrane insertion of tail-anchored proteins are known. The process
is ATP-dependent, consistent with a role for cytosolic
chaperones in maintaining the polypeptides in an integration-competent
state (19, 27-29). Furthermore, the membrane insertion of
tail-anchored proteins includes a membrane binding step that is
saturable (29), and this process is also sensitive to prior treatment
of the membranes with protease (19). Although both of these
observations suggest that the membrane insertion of tail-anchored
polypeptides is mediated by proteins, the ER components responsible for
this process have remained unidentified.
We set out to identify ER components that may mediate the membrane
insertion of tail-anchored proteins using a defined cross-linking approach to identify proteins that are transiently associated with the
newly membrane-integrated polypeptides. We report a defined sequence of
associations between newly made tail-anchored proteins and both the
Sec61 translocon and Sec61 translocon-associated components. Most
significantly, we observe a similar sequence of events with a
comparable, cotranslationally inserted, signal-anchored protein. We
conclude that the biosynthesis of tail-anchored and signal-anchored
proteins is mediated by a similar complement of ER components.
Antibodies--
Polyclonal antibodies were raised against
specific peptides representing Sec61 cDNA Constructs and Transcription--
An NcoI
fragment incorporating the coding region of the human Sec61 Translation and Membrane Insertion--
Proteins were
synthesized using rabbit reticulocyte lysate (Promega) that had been
prespun at 200,000 × g for 10 min to remove any
contaminating membranes. Incubations were performed at 30 °C in the
presence of both [35S]methionine and canine pancreatic
microsomes according to the manufacturer's instructions. Microsomes
were prepared from canine pancreas as described by Walter and Blobel
(31) and added to in vitro translations at 1.5-2.0
A280/ml. A time course was performed to
establish the amount of Sec61
Control experiments showed that in the absence of added microsomes, or
when using a protein that lacked its tail anchor sequence, less than
2% of the total protein synthesized in 30 min was recovered by this
assay (data not shown). In contrast, >30% of authentic Sec61 Cross-linking and Immunoprecipitation--
Microsomes were
isolated for cross-linking analysis by layering them over HSC buffer
(250 mM sucrose, 500 mM KOAc, 5 mM
Mg(OAc)2, 50 mM Hepes-KOH pH 7.9) and spinning
at 100,000 × g for 10 min to yield a membrane pellet.
Membrane pellets were resuspended in LSC buffer (250 mM
sucrose, 100 mM KOAc, 5 mM
(MgOAc)2, 50 mM Hepes-KOH pH 7.9) and normally
incubated immediately at 30 °C for 10 min with either
bismaleimidohexane (BMH) (0.5 mM final concentration unless
otherwise stated), diluted from a 20 mM stock dissolved in
dimethyl sulfoxide, or an equivalent dimethyl sulfoxide control. Where
time course experiments were carried out, the sample was resuspended in
LSC buffer and incubated at 30 °C for 0-120 min before
cross-linking as above. In one case a parallel sample was incubated at
0 °C for 60 min before adding BMH. Cross-linking was stopped by the
addition of 10 mM 2-mercaptoethanol to quench any unreacted
maleimide groups, and a fraction of the sample was removed for direct
analysis. The remainder of the samples were denatured at 70 °C for
10 min in the presence of 1% (w/v) SDS and diluted with 4 volumes of
Triton X-100 immunoprecipitation buffer (1% (w/v) Triton X-100, 140 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5). Samples were precleared by the addition of 10% volume Pansorbin (Calbiochem) and incubated at 4 °C for 1 h, followed by centrifugation at 15,000 × g for 5 min. The
resulting supernatants were subjected to immunoprecipitation by the
addition of antisera at 1:100 (v/v) and incubation at 4 °C for
16 h with mixing. Protein A-Sepharose was added to 10% volume,
and the incubation continued at 4 °C for a further 2 h. The
protein A-Sepharose beads and bound material were pelleted in a
microfuge and washed several times with Triton X-100
immunoprecipitation buffer. The resulting beads were heated to 70 °C
for 10 min in SDS-PAGE sample buffer, and unless otherwise stated, the
solubilized material was resolved on 16% polyacrylamide Tris-glycine
gels run under denaturing conditions.
Discrete Cross-linking Products Are Observed with Newly Synthesized
Sec61
Given our interest in the biogenesis of the ER translocon (32), we
chose Sec61
We then went on to compare samples of in vitro synthesized,
membrane-integrated Sec61
We analyzed the Sec61 The Majority of the Sec61
We therefore carried out a time course experiment where
membrane-associated Sec61
Interestingly, the "release" of newly synthesized Sec61
We did observe some reduction in the overall intensity of the
Sec61 Syb2 and Syn1A Associate with ER Membrane Components during
Membrane Integration--
To focus our analysis on potential
biosynthetic interactions, we next analyzed a tail-anchored protein
that would not be expected to form any stable complexes with endogenous
ER components. For this reason we chose Syb2 as a previously well
characterized tail-anchored protein that is membrane-integrated at the
ER and then transported to post-ER vesicular structures (19). When wild
type Syb2 was analyzed no BMH-dependent adducts were
observed, indicating that the single naturally occurring cysteine
residue present within the transmembrane domain of Syb2 (see Fig. 1) is
insufficient to generate BMH-dependent adducts (data not
shown). A number of previous studies have underlined the importance of
the relative position of a cross-linking probe within a precursor upon
its capacity for adduct formation (34). Given our success with the cross-linking analysis of Sec61
As we had seen previously with Sec61
To assess how general the associations of newly synthesized Sec61
An additional product was brought down nonspecifically during the
immunoisolation of the Sec61
On the basis of the data outlined above, we concluded that newly
synthesized tail-anchored proteins associate with several generic ER
components including the Syn1A Displays an Early and Transient Association with
Sec61a--
To address the possibility that tail-anchored proteins may
associate with distinct ER components at different stages during their
membrane insertion, we investigated the cross-linking partners of Syn1A
with respect to the time elapsed from the start of protein synthesis.
Syn1A was translated in the presence of microsomal membranes for only 5 min. Samples were then either placed on ice to stabilize transient
associations (Fig. 4, lanes 1-6; cf. Fig. 2C, lane 6) or "chased" at the translation
temperature for a further 15 min (Fig. 4, lanes 7-12),
prior to initiating BMH-dependent cross-linking. The most
telling result of this kinetic analysis of Syn1A cross-linking products
was our observation that the association between the newly synthesized
polypeptide and adjacent ER components is dynamic. Hence, we found
significant changes in the cross-linking partners between the two time
points studied. Most significantly, after a short 5-min incubation with
no chase, cross-linking of Syn1A to Sec61
Following a 15-min chase, during which no further protein synthesis
occurred, cross-linking to Sec61
The early association observed between Syn1A and Sec61 Tail-anchored and Signal-anchored Membrane Proteins Associate with
a Common Subset of ER Components--
To facilitate a direct
comparison between the post-translational membrane insertion of a
tail-anchored protein and the cotranslational insertion of a
signal-anchored protein, we chose to study the Ii of the human class II
major histocompatibility complex. Ii displays a short hydrophilic
N-terminal domain on the cytosolic side of the ER membrane (see Fig.
1), and its single transmembrane domain acts as the signal anchor
sequence that promotes its cotranslational insertion into the ER
membrane (36). To study the cotranslational insertion of Ii via its
signal anchor sequence, a transcript encoding the first 81 residues of
Ii, and lacking a stop codon (Fig. 1, Ii81), was translated in the
presence of ER derived microsomes. This leads to the resulting
ribosome-bound nascent chain being trapped at the ER insertion site
pending the puromycin-dependent release of the ribosome
from the nascent chain (37, 38).
The Ii was converted into a tail-anchored protein by introducing a stop
codon immediately after the end of the predicted transmembrane region
(Fig. 1, IiTA). Subsequent analysis of IiTA showed that it is indeed
capable of authentic post-translational membrane insertion and hence
behaves as a tail-anchored protein (data not shown). For both Ii81 and
IiTA the single naturally occurring cysteine residue close to the
cytosolic side of the predicted transmembrane region was substituted
for a methionine residue. To facilitate BMH-dependent
cross-linking, a single cysteine residue was then introduced into the
cytosolic domains of these polypeptides at a point 28 residues from the
start of the predicted transmembrane domain and hence at a location
comparable with the cysteine probe present in Sec61
As expected from previous studies (37, 38), the trapped form of Ii81
was efficiently cross-linked to Sec61
The cross-linking analysis of IiTA revealed a pattern of adducts which
resembled a combination of the results obtained with Ii81 in its
trapped and released forms (see Fig. 5), and adducts of IiTA with
Sec61
If the cross-linking of newly membrane-inserted IiTA to adjacent ER
components is acting to report a biosynthetic pathway faithfully, then
one would expect such associations to be transient as we had observed
for the majority of adducts detected with Sec61
The reduction in cross-linking efficiency which we do observe with
ribosome-bound Ii81 across a 60-min time course (Fig. 6B) is
most likely caused by the loss of the ribosomes from a proportion of
the Ii81 nascent chains during this incubation period (see Ref. 39). As
expected, if we released all of the chains from the ribosome at the
start of the time course by puromycin treatment, the nascent chains
were rapidly released from their association with the Sec61 During this study we have identified a discrete subset of cellular
components that are transiently associated with newly membrane-inserted tail-anchored proteins. Among these, we have identified five ER proteins that are either a part of the core ER translocon or closely associated with it. None of these ER components is specifically associated with tail-anchored proteins, and the same set of components is adjacent to a newly integrated signal-anchored membrane protein. Taken together, our data indicate that the pathway that facilitates the
membrane insertion of tail-anchored proteins displays substantial overlap with the "classical" pathway that mediates this process for
most other types of membrane proteins.
Newly Made Membrane Proteins Are in Transient Proximity to a
Specific Subset of ER Components--
All of the tail-anchored
proteins that we have studied display a transient association with a
defined subset of cellular components, as determined by site-specific
chemical cross-linking. A number of control experiments confirmed that
the time-dependent alterations in adduct formation which we
observe are a true reflection of changes in the local environment of
the newly integrated membrane proteins and not simply a loss of their
capacity to generate BMH-dependent adducts. We conclude
that, during their membrane insertion, newly synthesized tail-anchored
proteins move from a restricted environment with a high effective
concentration of specific ER proteins into a predominantly lipid
environment that reflects complete membrane integration (see Fig.
7).
Several cross-linking partners of the newly membrane integrated
tail-anchored proteins that we detect in our assay remain to be
identified. It is worth noting that at least some of these unidentified
components may represent transient interactions with cytosolic
components. Interactions between post-translationally translocated,
secretory protein precursors and cytosolic chaperones have previously
been detected in a rabbit reticulocyte lysate system (9). Furthermore,
in the case of tail-anchored membrane proteins the bulk of the
polypeptide remains on the cytosolic side of the membrane where it
would be available for such interactions even after membrane
integration. Interestingly, we also detect a far more complex pattern
of adducts with authentic tail-anchored proteins that have a
significant cytoplasmic segment than with our artificial tail-anchored
protein, IiTA, which does not (cf. Fig. 1).
On the basis of our cross-linking analysis we have been able to
identify several ER proteins that are transiently associated with newly
membrane-inserted tail-anchored proteins. In fact, by carrying out a
careful time course study of Syn1A integration, we could define
transient associations with two different sets of ER components that we
believe reflect different stages during the membrane insertion process.
We found that both wild type (Syn1A) and artificial (IiTA)
tail-anchored proteins are adjacent to Sec61 Role of the Sec61 Complex--
The Sec61 complex is known to play
a central role during the integration of a variety of cotranslationally
inserted membrane proteins (40-42) and acts as the principal gateway
for protein traffic across the ER membrane (43). We observe an early
association of tail-anchored proteins with both Sec61 Roles of Sec62p and Sec63p--
In this study, we show for the
first time that both Sec62p and Sec63p can be cross-linked to newly
synthesized membrane proteins in a mammalian cell free system. We also
find that the nature of the ER membrane insertion sequence,
i.e. tail-anchored or signal-anchored, has no influence upon
the transient proximity of a precursor to these components. Sec62p and
Sec63p are the mammalian equivalents of proteins that were first
identified in the yeast S. cerevisiae (15, 16). Yeast Sec62p
and Sec63p have long been associated with the post-translational
translocation of secretory proteins across the ER membrane (12-14),
although recent data suggest that Sec63p also functions during
cotranslational translocation (45). Consistent with this latter
proposal, we observe cross-linking to Sec63p at an early stage during
the cotranslational integration of a signal-anchored membrane protein.
Likewise, the interaction of tail-anchored proteins with mammalian
Sec63p is seen at an early time point during membrane insertion.
In yeast, Sec63p provides a binding site for the BiP ortholog, Kar2p,
and has been proposed to promote post-translational translocation in
addition to "gating" the Sec61 translocon at its luminal side (13,
14, 46). The functions of mammalian Sec62p and Sec63p remain to be
established (15, 16), but the proteins are present at roughly equimolar
amounts with respect to the Sec61 Proximity to SPC25--
The SPC25 subunit was identified as a
major cross-linking partner of all of the newly inserted membrane
proteins investigated during this study and was particularly apparent
at later stages of the membrane integration process. SPC25 is a subunit
of the mammalian signal peptidase complex, and it has been proposed to link the SPC complex to the Sec61 complex via the Sec61
In summary, our cross-linking analysis has identified ER components
that are associated with tail-anchored proteins during their membrane
insertion and shown that they closely mirror those associated with the
well characterized cotranslational pathway for membrane insertion. We
conclude that the pathways for the integration of tail-anchored and
signal-anchored membrane proteins at the ER exhibit a substantial
degree of overlap, and we believe that this reflects similarities
between the co- and post-translational pathways for membrane insertion.
The precise function of specific ER components during the membrane
insertion of tail-anchored proteins is the focus of our current work.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits, and it functions as the ER
membrane insertion site for precursors delivered via the
SRP-dependent targeting route (1-4). It is noteworthy that
in higher eukaryotes, the membrane insertion of proteins targeted via
the SRP-dependent pathway appears to be principally
cotranslational, with the ribosome remaining closely associated with
the Sec61 translocon during membrane integration (5, 6).
and Sec61
), vesicle recognition (soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor, or SNARE proteins), and electron transfer (cytochrome
b5) (for reviews, see Refs. 23 and 24). In the
case of synaptobrevin 2 (Syb2), the authentic membrane insertion
sequence can be replaced by a polyleucine span with a minimum length of
12 residues, demonstrating the lack of any specific sequence
requirements for this region (25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Sec61
(from B. Dobberstein), SPC25, Sec62p, and Sec63p. The anti-Syb2 antibody was a
mouse monoclonal recognizing an epitope at the N terminus of Syb2 (from
R. Jahn).
cDNA
(30) was subcloned into the pSPUTK vector (Stratagene). The DNA
template for its transcription was made by cleavage of the Sec61
pSPUTK plasmid with EcoRI. The cDNA for rat Syb2 in pBluescript was a gift from R. Scheller (Stanford University). The
cysteine mutant of synaptobrevin (Syb2cm) was created by changing a
leucine (Leu-63) to a cysteine using the QuikChange
site-directed mutagenesis kit (Stratagene). The following sense primer
was used: 5'-GACCAGAAGCTATCGGAATGCGATGATCGCGCAGATGCCC-3', and the
resulting plasmid was linearized for transcription using
XbaI. The cDNA for rat Syn1A was a gift from Dr. Sabine
Hilfiker (University of Manchester). The coding region was amplified by
PCR, introducing a BglII site 1 base 5' to the initiation
codon using primer 5'-ACTTTGGCAGATCTACCATGAAGGACCGAACCCAGG-3' and an
XbaI site after the termination codon using primer
5'-CACCATCGGGGGCATCTTTGGATAGTCTAGATATA-3'. This PCR product was cut
with BglII and ligated into the BglII and
HpaI sites of pSPUTK (Stratagene). The resulting plasmid was linearized for transcription using XbaI. The coding region
of invariant chain (Ii) was amplified by PCR using the forward primer 5'-ACTTTGGCAGATCTACCATGGATGACCAGCGCGACC-3' to introduce
BglII and NcoI sites around the initiation codon
and replace the asparagine residue at position 3 of the coding region
with a cysteine. An XbaI site was introduced after the
termination codon by using the reverse primer
5'-TATATCTAGATCACATGGGGACTGGGCC-3'. This PCR product was cut with
BglII and ligated into the BglII and
HpaI sites of pSPUTK (Stratagene). The natural cysteine
(Cys-28) was changed to a methionine using the QuikChange kit with the
sense primer 5'-CCGGAGAGCAAGATGAGTCGCGGAGCCCTG-3'. Transcription
templates for Ii derivatives were prepared by PCR using this variant of Ii as a template. The 5'-primer recognized a region 150 bp 5' to the
SP6 promoter 5'-CCAGAAACTCAGAAGGTTCG-3', whereas the 3' primers were
5'-TATATCAGTACAGGAAGTAGGCGGTGG-3' for Ii81 and 5'-CATGGGGACTGGGCCCAG-3' for IiTA. The authenticity of all PCR-derived constructs was
confirmed by DNA sequencing. Transcripts were synthesized using T3 RNA
polymerase for Syb2cm, or SP6 RNA polymerase for all the other
templates, according to the manufacturer's instructions (New England Biolabs).
that was integrated into
canine pancreatic microsomes under the experimental conditions that we were using. This analysis was carried out across a 60-min period, and
the percentage integration was defined as the proportion of the total
protein synthesized at a particular time point which was found to be
membrane-associated and resistant to extraction with alkaline sodium
carbonate solution (23, 32).
was
recovered in the membrane fraction using the same assay (see Fig.
2A). On the basis of this analysis, translation reactions were initially carried out for 30 min by which point significant membrane integration was observed (see Fig. 2A). In some
subsequent experiments, shorter incubation periods were used to
optimize transient associations. In these cases, specific details are
indicated in the accompanying figure legend. All puromycin treatments
were performed by the addition of 1 mM puromycin and
subsequent incubation at 30 °C for 5 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
We chose to investigate the association of newly
synthesized tail-anchored proteins with ER components using the
bifunctional cross-linking reagent BMH, which is highly specific for
cysteine residues. This approach had proven very efficient in previous studies of cotranslational membrane protein biosynthesis (32, 33).
Furthermore, our preliminary analysis had shown that the membrane
insertion of the tail-anchored proteins into ER-derived microsomes
could be prevented by prior treatment of the membranes with
N-ethylmaleimide, which modifies the free sulfydryl groups of cysteine residues (data not shown). Hence, BMH was ideally suited to
cross-link newly synthesized tail-anchored proteins containing one or
more cysteine residues (see Fig. 1) to
the N-ethylmaleimide-sensitive, cysteine-containing ER
proteins that facilitate their membrane insertion.
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Fig. 1.
Proteins used for membrane insertion
studies. The proteins are annotated to show their membrane
topology, the number of amino acid residues (aa) in each
putative domain, and the approximate location of cysteine residues.
Black circles represent engineered cysteines, and gray
circles represent natural cysteines. Sec61 , with a single
cysteine residue located 32 residues from the start of the predicted
transmembrane, Syb2cm, with an additional cysteine positioned 32 residues from the start of the predicted transmembrane, and Syn1A, with
a cytosolic cysteine positioned 121 residues from the start of the
predicted transmembrane, are naturally occurring tail-anchored
proteins. Ii was engineered to possess a single cysteine 28 residue
from the start of the putative transmembrane. Ii was synthesized in
truncated forms of 81 residues lacking a stop codon (Ii81) and in
tail-anchored form comprising 56 residues with a stop codon
(IiTA).
, one of the tail-anchored subunits of the Sec61 complex,
for our initial studies. The
and
subunits of the Sec61 complex
both have amino acid sequences that are characteristic of tail-anchored
proteins (30) and, like Syb2 (19), are capable of authentic
post-translational membrane integration (data not shown). A preliminary
time course experiment with Sec61
showed that during a 30-min
incubation in the presence of canine pancreatic microsomes, more than
30% of the total protein synthesized was both membrane-associated and
resistant to extraction with alkaline sodium carbonate solution,
indicating that it was fully integrated (Fig.
2A; see also
"Experimental Procedures" and Refs. 23 and 32).
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Fig. 2.
Membrane integration of
Sec61 analyzed by BMH-dependent
cross-linking. A, Sec61
was synthesized in the presence
of canine pancreatic microsomes for 10, 30, or 60 min, and the
membrane-associated fraction was isolated by centrifugation through a
high salt cushion as described under "Experimental Procedures."
This fraction was extracted with alkaline sodium carbonate buffer, and
the reisolated membrane pellet was analyzed by SDS-PAGE in parallel
with a sample of the total protein synthesized during the same period
of time. The percentage of membrane integration indicates the
proportion of the total protein synthesized at a particular time point
which was resistant to extraction with alkaline sodium carbonate
solution (cf. Ref. 32). B, Sec61
was
translated in the presence of microsomes for 30 min, the
membrane-associated fraction was isolated, and samples were then treated with or without BMH as indicated. Total products
(lanes 1 and 2) or products immunoprecipitated by
specific antisera (lanes 3-8) were then analyzed by
SDS-PAGE as indicated. A filled arrowhead marks the position
of the Sec61
precursor protein. Adducts with specific proteins are
indicated with the following symbols:
, Sec61
;
, SPC25; and §,
Sec63p. The mobility of the Sec61
adduct is distorted in lanes
3 and 4 by the presence of comigrating IgG heavy chain.
Unidentified adducts of ~22 (
) and ~40 kDa (×) are also
indicated. C, newly synthesized, membrane-associated
Sec61
was incubated at 30 °C for increasing lengths of time
(0-120 min) before cross-linking with 1 mM BMH
(lanes 1-5). In one case, a sample of the
membrane-associated Sec61
was placed on ice immediately and
incubated at 0 °C for 60 min before cross-linking with 1 mM BMH (lane 6). After quenching, the samples
were extracted with alkaline sodium carbonate solution, and the
membrane fraction was reisolated and analyzed by SDS-PAGE on a 15%
polyacrylamide gel. Major cross-linking products are indicated as in
B. A filled arrowhead indicates the location of
the Sec61
precursor.
incubated in the presence and absence of
BMH and discovered a number of specific cross-linking products (Fig.
2B, lanes 1 and 2). In particular,
prominent adducts of ~22, ~34, ~40, and ~50 kDa were seen (Fig.
2B, lane 2), suggesting the cross-linking of the
~12-kDa Sec61
polypeptide chain to cellular components of ~10,
~22, ~28, and ~38 kDa, respectively. It should be noted that the
apparent mobility of cross-linking products on SDS-PAGE might not
accurately reflect the sum of the molecular masses of the cross-linked
components. Hence, in the absence of additional information the
predicted sizes of the cross-linking partners must be treated with caution.
cross-linking products by immunoprecipitation
using a variety of antisera to ER components and were able to identify
two of the major adducts unambiguously. Thus, newly synthesized
membrane-integrated Sec61
was found to be cross-linked to the
subunit of the Sec61 complex (Fig. 2B, lane 3)
and SPC25, the 25-kDa subunit of the signal peptidase complex (Fig.
2B, lane 5). As expected, all of the adducts were
immunoprecipitated by an antiserum recognizing Sec61
(Fig.
2B, lane 4). Furthermore, cross-linking of
Sec61
to both Sec62p and Sec63p was also observed (Fig.
2B, lanes 6 and 7, respectively),
although these did not appear to be major adducts (Fig. 2B,
lanes 2 and 4).
Cross-linking Products Reflect
Transient Associations--
Given that Sec61
forms part of the
heterotrimeric Sec61 complex, we reasoned that newly synthesized,
membrane-inserted Sec61
would probably display two types of
associations with endogenous ER components. These would most likely be
transient associations, indicative of a biosynthetic pathway, and
stable associations, indicative of assembly into the Sec61 complex.
was isolated from the translation reaction and incubated for increasing periods of time before cross-linking was
initiated. From this time course experiment, it was immediately obvious
that all of the major cross-linking products except the Sec61
adduct
reflected a transient association between the newly synthesized
Sec61
polypeptides and ER-associated components (Fig. 2C,
lanes 1-5; cf. Fig. 2B, lane
3). Thus, the pattern of BMH-dependent cross-linking
products was the same as observed previously when cross-linking was
carried out immediately after the isolation of the membrane fraction
(Fig. 2B, lane 2, and Fig. 2C,
lane 1). In contrast, when the membrane fraction was
incubated for 10 min at the translation temperature before
cross-linking, only the prominent Sec61
adduct remained (Fig.
2C, lane 2, product indicated by the filled
circle in lane 6). The sample could be incubated at
30 °C for up to 120 min before adding the cross-linking reagent, and
a strong adduct with Sec61
was still observed, whereas none of the
other major products seen at the zero time point was detected (Fig.
2C, lanes 1-5).
from its
transient association with a discrete set of cellular components could
be prevented by incubating the samples on ice rather than at 30 °C
(Fig. 2C, lanes 4 and 6). This
observation indicates that the release of Sec61
from these transient
associations is prevented at low temperature. We found that adduct
formation with Sec61
was largely unaffected by prolonged incubation
before initiating BMH-dependent cross-linking, confirming
the stability of reduced thiol groups under the experimental conditions
we have used. The prolonged association of Sec61
with Sec61
which
we can detect by cross-linking presumably reflects the in
vitro formation of Sec61-derived complexes.
/Sec61
adduct across a 120-min time course, although the
radiolabeled Sec61
chains were stable during this period (Fig.
2C, lanes 1-5). In contrast, the
Sec61
/Sec61
adduct was unaffected by a 60-min incubation at
0 °C (Fig. 2C, lanes 4 and 6). We
believe that Sec61
can be cross-linked to Sec61
both during its
biosynthesis, where the association is transient, and during subsequent
complex formation, where the association is stable. In the case of
complex formation, the radiolabeled Sec61
synthesized in
vitro must compete for binding with endogenous unlabeled Sec61
present in the canine pancreatic microsomes. Such competition would
limit the proportion of radiolabeled Sec61
that could be
cross-linked to Sec61
, and presumably a "steady state" is
reached. This is also consistent with our observation that between 30 and 120 min the efficiency of cross-linking to Sec61
is relatively
constant (Fig. 2C, lanes 3-5). On the basis of
our cross-linking analysis with Sec61
, we concluded that
BMH-dependent cross-linking could be exploited successfully
to reveal transient associations between newly synthesized
tail-anchored proteins and cellular components.
, we prepared a mutant form of Syb2
with a cysteine residue at an equivalent location within its cytosolic
domain (Syb2cm, see Fig. 1). We confirmed that Syb2cm behaved as an
authentic tail-anchored protein by showing that it could integrate
post-translationally into canine pancreatic microsomes in the same way
as the wild type protein (cf. Ref. 19 and data not shown).
(Fig. 2, B and
C), Syb2cm yielded several BMH-dependent
cross-linking products when synthesized in the presence of ER-derived
microsomal membranes (see Fig.
3A, lanes 1 and
2). A parallel control experiment showed that no such
BMH-dependent adducts were obtained with a version of
Syb2cm which lacked its transmembrane domain (data not shown). All of
the BMH-dependent adducts could be immunoprecipitated with an anti-Syb2 antibody confirming their origin (Fig. 3A,
lane 3). By screening a number of antisera recognizing known
ER components, we were able to identify specific cross-linking of
Syb2cm with Sec61
(~10-kDa partner, Fig. 3A, lane
5, *) and SPC25 (~22-kDa partner, Fig. 3A, lane
6,
). The ~38-kDa product observed in the absence of BMH (Fig.
3A, lane 1, thin arrow) is
distinct from the SPC25 adduct (Fig. 3A, lane 6)
and is most likely an SDS-resistant dimer of Syb2cm. A weaker ~80 kDa
adduct with Sec62p (Fig. 3A, cf. lanes 7 and
8) could also be observed after immunoprecipitation. All three of these components are known to be either a part of, or
closely associated with, the Sec61 translocon. Despite this, we could
detect no cross-linking of Syb2cm to Sec61
, the core subunit of the
Sec61 translocon (Fig. 3A, lane 4). This is in contrast to the behavior of newly synthesized Sec61
, which exhibited strong cross-linking to Sec61
which generated a discrete ~50-kDa adduct (see Fig. 2B). The identity of at least three other
major Syb2cm cross-linking products remains to be determined (see
"Discussion").
View larger version (107K):
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Fig. 3.
Syb2cm and Syn1A associate with
translocon-associated components. Syb2cm (A) and Syn1A
(B) were translated in the presence of microsomes for 30 min, treated with or without BMH, and then immunoprecipitated
(IP) with various antibodies as indicated. Filled
arrowheads mark the positions of the precursor protein. In
lanes 1 and 2 (in A) and lane
1 (in B) the products were not subjected to
immunoprecipitation. In lane 7 (in A) the
preimmune serum (PI) matched to the anti-Sec62p serum was
used as a control. The products were separated by SDS-PAGE, and the
specific cross-linking products are indicated with the following
symbols: *, Sec61 ; and
, SPC25. In both cases, several strong
adducts remain unidentified (see A, lane 3, and
B, lane 1); with Syn1A, traces of one product
appeared nonspecifically after immunoprecipitation (B,
lanes 3-5, product marked by an open arrowhead).
In A, lane 1, a thin arrow indicates a
putative SDS-resistant Syb2cm dimer.
and Syb2cm with specific ER components were, we repeated a similar
experiment using a third tail-anchored protein. Syn1A has only one
cysteine present in its cytosolic domain, and this is located 121 residues from the presumptive transmembrane region (Fig. 1).
Nevertheless, like the other two proteins analyzed, Syn1A also yielded
multiple BMH- dependent cross-linking products (Fig.
3B, lane 1). As with Syb2cm, specific adducts of
Syn1A with Sec61
(Fig. 3B, lane 3, *),
SPC25 (Fig. 3B, lane 4,
), and Sec62p (Fig.
3B, lane 5, ~90 kDa product) were
identified. At least two other major adducts were detected (Fig.
3B, lane 1; Fig. 4,
lane 7), but these could not be identified by
immunoprecipitation (see "Discussion").
View larger version (115K):
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Fig. 4.
Syn1A is associated with
Sec61 early and transiently. Syn1A was
translated in the presence of microsomes for 5 min, then either held on
ice or treated with puromycin and incubated at 30 °C for a further
15 min (chase). Samples were then treated with BMH and
immunoprecipitated with various antibodies as indicated. A filled
arrowhead marks the position of the precursor protein. Lanes
1 and 7 show the total products prior to
immunoprecipitation (
). Products were separated by SDS-PAGE, and
specific cross-linking products are indicated with the following
symbols:
, Sec61
; *, Sec61
;
, SPC25; and §,
Sec63p.
, SPC25, and Sec62p adducts (Fig.
3B, lanes 3, 4 and 5,
open arrowhead). However, in other experiments this product
was not associated with these adducts after immunoprecipitation
(cf. Fig. 4.). Once again, Syn1A showed no evidence of
cross-linking to Sec61
(Fig. 3B, lane 2). The authenticity of the Syn1A adducts with Sec61
, SPC25, and Sec62p was
confirmed further by showing that no such products could be detected
when control immunoprecipitations were carried out in the absence of
BMH-dependent cross-linking (data not shown).
subunit of the Sec61 complex and two Sec61
associated components (SPC25 and Sec62p). In contrast to many previous
studies that had investigated cotranslationally inserted membrane
proteins (see Ref. 2 and references therein), no cross-linking of the
tail-anchored precursors to Sec61
was observed. We reasoned that
this may be the result of any one of several factors: 1)
Sec61
does not associate with tail-anchored proteins during their
membrane integration; 2) Sec61
associates at an earlier stage of the
process than we had analyzed; 3) the cysteines present in Sec61
were
unsuitable for BMH-dependent cross-linking to the
tail-anchored proteins we had used in this study. Our previous success
with BMH-dependent cross-linking (32, 33), coupled with our
ability to cross-link Sec61
to Sec61
in the context of complex
formation (see Fig. 2, B and C), suggested that
the third possibility was unlikely. We therefore investigated whether
the ER components that are adjacent to newly synthesized tail-anchored
proteins are in any way dependent upon the relative time at which
cross-linking is carried out.
was detected (Fig. 4,
lane 2,
). The other characteristic of this "early"
stage of Syn1A integration was a very large adduct that included
mammalian Sec63p (Fig. 4, lane 6, §). The difference between the predicted (~120 kDa), and apparent (~180 kDa) size of
this adduct (Fig. 4, lane 6, §) suggests that either its
mobility is aberrant, or the adduct contains one or more components in addition to Syn1A and Sec63p (cf. Ref. 15). At this early
stage, weak adducts with Sec61
, SPC25, and Sec62p were also detected (Fig. 4, lanes 3-5).
and Sec63p was lost (Fig. 4,
lanes 8 and 12), whereas the other adducts were
retained and/or showed an increase in intensity (Fig. 4, lanes
9-11). The presence of Syn1A adducts with Sec62p and SPC25 even
after only 5 min of protein synthesis most likely reflects our
inability to generate artificially trapped "integration
intermediates" of the tail-anchored proteins studied (cf.
Ref. 33). Thus, even after a relatively short period of synthesis, a
spread of molecules representing different stages of the membrane
integration process will be present in the reaction mixture. Given this
experimental limitation, the simplest interpretation of these results
is that Syn1A is transiently associated with Sec61
and Sec63p at an
early stage of the membrane insertion process. Adducts of Syn1A with Sec61
are also observed at this early stage of synthesis; however, in contrast to Sec61
and Sec63p this association is more prolonged (Fig. 4, cf. lanes 3 and 9). The
proximity of Syn1A to SPC25 and Sec62p occurred predominantly at
"later" stages of the membrane integration process, i.e.
after a 15-min chase (Fig. 4, cf. lanes 4 and
5 with lanes 10 and 11).
suggests that
Syn1A may be associated with the Sec61 translocon at an early stage of
its membrane insertion. This conclusion is supported by our observation
of downstream interactions between Syn1A and Sec61-associated
components, specifically Sec62p and Sec61
/SPC 25 (see Ref. 35,
cf. Refs. 15 and 16). Given that no other integral membrane
proteins had previously been shown to associate with Sec62p and Sec63p
during membrane insertion, it was possible that these components were
specific for tail-anchored proteins.
and Syb2cm (see
Fig. 1).
(Fig.
5, lane 2, filled
circle). In addition, we observed adducts of Ii81 with Sec61
,
SPC25, and Sec63p (Fig. 5, lanes 3, 4, and
6), none of which had been identified during previous
studies that employed alternative cross-linking strategies
(cf. Refs. 37 and 38). When samples were treated with
puromycin before cross-linking, the adducts with Sec61
and Sec63p
were almost completely lost (Fig. 5, cf. lanes 2,
6, 8, and 12). Cross-linking to
Sec61
was also diminished (Fig. 5, lane 9), whereas
adducts with both SPC25 and Sec62p were enhanced (Fig. 5, lanes
10 and 11).
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Fig. 5.
Tail-anchored proteins associate with the
same ER proteins as those that are signal-anchored.
Left, Ii81 was translated in the presence of
microsomes for 10 min, treated with BMH, and immunoprecipitated with
various antibodies. The samples were resolved by SDS-PAGE, and specific
cross-linking products are marked with the following symbols: ,
Sec61
; *, Sec61
;
, SPC25; and §, Sec63p. An
arrowhead indicates the precursor protein.
Center, same as left panel but with a 5-min
puromycin treatment after translation. Right, same as
left panel but with a 5-min translation of IiTA.
, Sec61
, SPC25, Sec62p, and Sec63p could be observed (Fig.
5, lanes 14-18). Hence, the tail-anchored form of Ii
associates with the same group of ER components as the authentic tail-anchored proteins used during this study (cf. Figs.
2-5). More significantly, the ER components that we have shown to
associate with three tail-anchored membrane proteins during their
membrane insertion are identical to those associated with a well
characterized signal-anchored protein that is delivered to the ER
membrane via the cotranslational pathway.
(cf. Fig.
2C). We therefore carried out a detailed time course study
of the cross-linking of IiTA to known ER components. This analysis
showed that association of IiTA with all five of the ER proteins that
we could identify by immunoprecipitation was transient and that the
proximity of IiTA to Sec61
was particularly short lived (Fig
6A). Our studies of Sec61
integration had shown that the efficiency of its cysteine-mediated
cross-linking to Sec61
was not reduced dramatically by prolonged
incubation times and not reduced at all if samples were held at 0 °C
(cf. Fig. 2C). These data indicated that the
inactivation of cysteine residues by oxidation was not responsible for
the time-dependent loss of cross-linking which we observed
with IiTA. Nevertheless, we also addressed this issue with Ii by taking
advantage of its cotranslationally inserted form (Ii81). When a
ribosome-bound integration intermediate of Ii81 was generated and
treated with BMH across a time course identical to that used to study
IiTA, we found that adduct formation was relatively stable in all cases
(Fig. 6B). Thus, we can conclude that the loss of
cross-linking to IiTA which we observe across the same time course
cannot be solely the result of the loss of available cysteine
residues.
View larger version (52K):
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Fig. 6.
Time course of Ii81 and IiTA cross-linking to
membrane components. A, IiTA was translated in the presence
of microsomes for 10 min and treated with puromycin for 10 min. The
microsomes were isolated, then incubated at 30 °C for an additional
0, 20, or 50 min, and finally treated with BMH and immunoprecipitated
with various antibodies. The resulting adducts were separated by
SDS-PAGE and quantified such that each adduct was 100% at its highest
value over the time course. The time scale begins at the addition of
puromycin. B, Ii81 was translated in the presence of
microsomes for 10 min. The microsomes were isolated, then incubated at
30 °C for an additional 0 or 60 min, and finally treated with BMH
and immunoprecipitated with various antibodies. The resulting adducts
were separated by SDS-PAGE and quantified such that each adduct was
100% before incubation. The time scale indicates the post-translation
incubation. C, Ii81 was translated in the presence of
microsomes for 10 min and treated with puromycin for 10 min. The
microsomes were isolated, then incubated at 30 °C for an additional
0, 20, or 50 min, and finally treated with BMH and immunoprecipitated
with various antibodies. The resulting adducts were separated by
SDS-PAGE and quantified such that each adduct was 100% at its highest
value over the time course. The time scale begins at the addition of
puromycin, and the value at time zero was taken from B,
therefore representing the ribosome-arrested state.
and
Sec61
subunits of the core ER translocon (Fig. 6C). In
contrast, the puromycin-released chains remained adjacent to Sec62p and
SPC25 for a longer period, although this association was still clearly
transient (Fig. 6C). We therefore conclude that the co- and
post-translational pathways for the insertion of newly synthesized
membrane proteins at the ER utilize a similar subset of ER components.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Schematic view of the ER membrane insertion
site. The Sec61 translocon is depicted from a top view
based on known relationships among specific components (see the
Introduction). The filled black circles represent the
proposed pathway taken by a newly inserted tail-anchored protein during
its integration into the phospholipid bilayer. Tail-anchored proteins
interact early and transiently with Sec61 , the major component of
the Sec61 complex, and Sec63p is also implicated at an early stage
during integration. Interactions with Sec61
occur at both early and
late stages, whereas SPC25 and Sec62p are principally involved at later
stages of integration. Newly synthesized proteins with a signal anchor
sequence display similar associations, although the interaction with
Sec61
is maintained during synthesis of the ER luminal domain and
can be artificially stabilized by using ribosome-bound integration
intermediates. Clearly, this model does not preclude a role for
additional, as yet unidentified, components during the membrane
integration of tail-anchored proteins.
and Sec63p at an early
stage during their membrane insertion. The proximity of the newly
membrane-inserted chains to these components is brief and is lost after
a short chase period. In contrast, the cross-linking products that we
observed with SPC25 and Sec62p were more prevalent at later time points
consistent with associations at a later stage of the membrane insertion
process. As already indicated, our inability to generate integration
intermediates of tail-anchored membrane proteins undoubtedly limits the
precision of this type of "stage specific" cross-linking analysis.
However, we found that a comparable cotranslationally inserted membrane protein (Ii81) associated with the same ER components and showed a
similar time dependence when released from its ribosome-arrested state.
Hence, the ribosome-bound form of Ii81 was predominantly cross-linked
to Sec61
, Sec61
, and Sec63p as well as some adduct formation with
SPC25. The release of the ribosome from the trapped integration
intermediate led to the loss of the Sec63p adduct and a large reduction
in cross-linking to Sec61
combined with an increase in cross-linking
to SPC25 and the appearance of an adduct with Sec62p. Thus, whatever
the precise role of these five components during membrane insertion, we
can conclude that none of them appears to be specifically associated
with tail-anchored proteins.
and Sec61
,
suggesting that the Sec61 complex might play some role during the
membrane insertion of tail-anchored proteins (see Fig. 7). This
proposal is consistent with the role of Sec61 in the post-translational translocation of small secretory proteins in higher eukaryotes (44). It
is noteworthy that the purified Sec61 complex alone is incapable of
supporting the membrane insertion of tail-anchored proteins (19). Our
cross-linking data identify additional components including Sec62p and
Sec63p, and such proteins may represent essential upstream elements of
the pathway for tail-anchored membrane protein integration which were
lacking from the purified system previously used by Kutay et
al. (19). At present we can only speculate about the role played
by the Sec61 translocon during the biosynthesis of tail-anchored
proteins. The simplest model to explain our data is that the Sec61
complex is the actual site of tail-anchored protein insertion into the
ER membrane. Alternatively, the interaction of newly made tail-anchored
proteins with translocon-associated components such as Sec62p or Sec63p
may account for their transient proximity to the Sec61 complex.
monomer. Consistent with our
cross-linking data, a fraction of Sec62p and Sec63p is clearly
associated with the Sec61 complex (15, 16). We find membrane proteins
are adjacent to Sec63p at an early stage during their insertion (Fig.
7), and this is consistent with a role for Sec63p, perhaps together
with BiP, in regulating or priming the ER translocation site (13, 16,
47). In contrast, we find that newly made polypeptides are adjacent to
Sec62p at a late stage of the membrane integration process. Thus,
Sec62p presumably facilitates a different stage of the process, for
example the transition of the transmembrane domain from the Sec61
complex into the phospholipid bilayer (see also Ref. 11).
subunit (35). The physical proximity between newly synthesized tail-anchored proteins and the signal peptidase complex which we detect by
cross-linking is in close agreement with recent evidence for a
functional interaction (48). Thus, when a consensus site for signal
peptidase cleavage is introduced at the C terminus of Syb2, derivatives
with a sufficiently long transmembrane spanning region are
authentically processed by the signal peptidase complex (48). The fact
that we identify SPC25 as a major cross-linking partner of all newly
made membrane proteins analyzed, both tail-anchored and
signal-anchored, indicates that such polypeptides may be scanned by the
signal-peptidase complex during their exit from the ER membrane
insertion site. This process would ensure the efficient recognition and
processing of cleavable ER targeting signals where present (see Fig. 7
and Ref. 2).
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ACKNOWLEDGEMENTS |
---|
We thank Richard Scheller for the synaptobrevin construct and Sabine Hilfiker for the Syn1A cDNA. We thank Bernhard Dobberstein and Reinhard Jahn for supplying antisera and monoclonal antibodies. Many thanks to Viki Allan, Neil Bulleid, Sam Crawshaw, and Sabine Hilfiker for invaluable help with the preparation of the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC), the Deutsche Forschungsgemeinschaft, the European Union, and the Medical Research Council and by a BBSRC professorial fellowship (to S. H.).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.
§ Present address: Research Toxicology Section, Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, U. K.
¶ Present address: Tissue Engineering Centre, Faculty of Medicine, Imperial College, 369 Fulham Rd., London SW10 9NH, U. K.
To whom correspondence should be addressed. E-mail:
stephen.high@man.ac.uk.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M209968200
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ABBREVIATIONS |
---|
The abbreviations used are: SRP, signal recognition particle; BMH, bismaleimidohexane; ER, endoplasmic reticulum; Ii, invariant chain; SPC25, 25-kDa subunit of the signal peptidase complex; Syb2, synaptobrevin 2; Syb2cm, cysteine mutant of synaptobrevin 2; Syn1A, syntaxin 1A.
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