Duke University Medical Center, Department of Cell Biology, Durham, North Carolina 27710
Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.
In mammalian cells, the translocation of nascent chains
across the endoplasmic reticulum (ER) membrane is
obligatorily cotranslational, and is thought to take
place through an aqueous channel composed primarily of
the resident ER membrane protein Sec61p and, in some cases, TRAM (Görlich and and Rapoport, 1993; Mothes
et al., 1994 SEC61 was discovered in a genetic screen designed to
identify components of the yeast protein translocation pathway, and encodes a polytopic 54-kD ER membrane protein (Deshaies and Schekman, 1987 That there exists within the rough ER a specific machinery dedicated to ribosome binding is embodied in current
models of translocation (Görlich et al., 1992 Extensive characterization of the ribosome-membrane
junction has established that binding is mediated in part by
the nascent chain and by protease-sensitive, electrostatic
interactions between the ribosome and components of the
ER membrane (Adelman et al., 1973 Three candidate ribosome receptors, p180, p34, and the
Sec61p complex, have been identified by the ribosome
binding protocol of Borgese et al. (1974) There is substantial experimental evidence in support of
a ribosome receptor function for the Sec61p complex.
Upon detergent solubilization and centrifugation of rough
microsomes (RM), Sec61p was found in the ribosomeenriched pellet fraction, along with a subset of other ribosome-associated membrane proteins, or RAMPS (Görlich
et al., 1992 Using established criteria for differentiating membrane
bound vs free ribosome/nascent chain complexes, we report that the binding of translationally active ribosome/
nascent chain complexes to the ER membrane is insensitive to the ribosome occupancy state of the Sec61p complex, and is not blocked by addition of a large molar excess
of free 80 S ribosomes. However, and consistent with previous reports, we observed that of the identified ribosome receptors, only Sec61p was protected from proteolytic
digestion by native, bound ribosomes. To reconcile this
apparent paradox, we evaluated the hypothesis that the
binding of active, nascent chain bearing ribosomes is comprised of Sec61p independent and Sec61p dependent
stages. When binding reactions were performed with limiting concentrations of RM, and thus limiting levels of ribosome-unoccupied Sec61p, bound nascent chains, although
protease resistant, were sensitive to extraction with high
salt, thereby identifying a novel state of nascent chain association with the ER membrane. With nonlimiting concentrations of RM, bound nascent chains were protease
and salt resistant. In related experiments, it was observed
that the efficiency of nascent chain cross-linking to Sec61p
was a function of RM concentration. Thus, at limiting RM
concentrations the yield of nascent chain/Sec61p crosslinks was markedly reduced relative to that observed at
nonlimiting RM concentrations. On the basis of these data,
we propose that ribosome/nascent chain association with
the ER membrane is a multi-stage process comprised of an
initial Sec61p independent and a subsequent Sec61p dependent stage.
Reagents
Hemin, creatine phosphate, and creatine phosphokinase were obtained
from Calbiochem (San Diego, CA). Staphylococcal nuclease, calf liver tRNA,
puromycin, and proteinase K were obtained from Boehringer Mannheim
Biochemicals (Indianapolis, IN). Phenylhydrazine hydrate and trypsin was
from Sigma Chem. Co. (St. Louis, MO). Chymotrypsin was from Worthington Scientific Corporation (Freehall, NJ). Restriction enzymes were
obtained from either New England Biolabs (Beverly, MA) or Promega
(Madison, WI). [35S] Pro-Mix ([35S] methionine and cysteine) was obtained
from Amersham (Arlington Heights, IL). Nucleotides were obtained from
Pharmacia (Piscataway, NJ).
Membrane Protein Protease Accessibility
Protease accessibility studies in canine and porcine RM was performed as
follows: four equivalents (eq.) of RM were diluted in a buffer containing
25 mM K-Hepes, pH 7.2, 25 mM KOAc, and 2.5 mM Mg(OAc)2 to a final
volume of 100 µl. Chymotrypsin was added to the indicated concentrations from a 1-mg/ml stock solution. Protease digestions were performed
for 30 min at 4°C. After digestion, samples were precipitated by addition
of TCA to a final concentration of 10%, and processed for SDS-PAGE.
Transfer to nitrocellulose membranes for immunoblot analysis was performed by semi-dry transfer in a 50 mM CAPS, pH 11.0, 20% methanol,
0.075% SDS buffer. Immunoblots were visualized by ECL detection (Amersham Corp.). Immunoblot films were scanned on a Hewlett-Packard
Scanjet Plus, and size and contrast adjusted in Photoshop version 3.0 (Adobe Systems, Inc., Mountain View, CA). Quantitation of imaged immunoblots was by means of NIH Image software.
Generation of Anti-Ribosomal Antibodies
Ribosomes were prepared from deoxycholate treated canine RM by the
method of Florini and Breuer (1966) Cell-Free Transcription and Translation
The plasmid pGEMBP1 (Connolly and Gilmore, 1986 Quantitation of Cell-Free Translation Products
The amount of free, nonradioactive methionine in the cell-free translation
system was determined by isotope dilution. The addition of 1.4 µM nonradioactive methionine to the translation system decreased incorporation of
radioactive methionine by 50%. Therefore, calculations of translation
yield were based on an endogenous methionine concentration of 1.4 µM.
The specific activity of the methionine pool, expressed as PSU units/nmol,
was determined by phosphorimager based quantitation of a serial dilution
series of the translation mix. The contribution of isotopically labeled cysteine to the total radioactivity of the translation products was <5% and
was not included in the calculation.
Preparation of EDTA and KOAc Washed RM (EKRM)
EKRM were prepared by diluting 250 eq. of RM fourfold in buffer to yield
final concentrations of 0.5 M KOAc, 10 mM EDTA, 25 mM K-Hepes, pH
7.2. After a 30-min incubation at 4°C, the membranes were collected by
centrifugation for 10 min at 60,000 rpm in a TLA 100.2 rotor at 4°C (Beckman Instrs., Fullerton, CA). EKRM pellets were resuspended in RM
buffer (0.25 M sucrose, 25 mM K-Hepes, pH 7.2, 25 mM KOAc), and
stored at Reconstitution of SR Isolation, partial purification, and reconstitution of the 52-kD fragment of
SR Sec61p Purification and Quantitation
Sec61p was purified by a modification of the procedures of Görlich and
Rapoport (1993) Purification of Reticulocyte Ribosomes
8 ml of nuclease-treated reticulocyte lysate was diluted to 12 ml using ribosome buffer (150 mM KCl, 25 mM K-Hepes, pH 7.2, 5 mM Mg(OAc)2).
Lysate was then centrifuged for 35 min at 100,000 rpm in the TLA100.3
rotor (4°C). The supernatant was removed by aspiration, and the pellet resuspended in 1 ml of ribosome buffer by Dounce homogenization (B pestle) and agitation for 2 h at 4°C. Insoluble material was removed by centrifugation for 10 min at 10,000 g. Samples were then loaded on
preparative 10-30% sucrose gradients and centrifuged at 40,000 rpm for 2 h
at 25°C in the SW40.1 rotor. The lower 50% of each gradient was collected, combined, and centrifuged for 3 h at 45,000 rpm in the Ti50.2 rotor,
4°C. The supernatant was aspirated, the 80-S ribosomal pellets resuspended in ribosome buffer, and aliquots stored at NEM Treatment of RM
RM were diluted fivefold and treated with 1 mM NEM (200 mM stock in
DMSO) for 20 min at 25°C. After treatment, DTT was added to a final
concentration of 25 mM, and reactions incubated for an additional 10 min
at 25°C. RM were layered over 0.5 M sucrose cushion, and collected by
centrifugation (6 min, 60,000 rpm, TLA 100 rotor, 4°C). RM pellets were
resuspended in RM buffer supplemented with 2 mM DTT.
Chemical Cross-linking
Chemical cross-linking of completed translation reactions were performed
as follows. Translation reactions were chilled on ice and diluted fivefold
with a physiological salt buffer consisting of 110 mM KOAc, 25 mM K-Hepes
(pH 7.4), 2.5 mM Mg(OAc)2. Diluted reactions were overlayed onto a 1/3
vol cushion of 0.5 M sucrose, 25 mM K-Hepes, pH 7.4 and centrifuged for
10 min at 60,000 rpm in the TL100 rotor. Supernatant and cushion fractions were discarded and the membrane pellet resuspended in 0.25 M sucrose, 50 mM KOAc, 2.5 mM Mg(OAc)2. Cross-linking reactions (50 µl)
were performed for the indicated time periods at 25°C, by addition of
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to a final concentration of 1 mM, from a 50-mM stock in dimethylformamide. Reactions were quenched by addition of 1 vol of PBS containing 50 mM dithiothreitol, 50 mM lysine, 1% SDS. Cross-linking reactions were precipitated
by addition of TCA to 10% and processed for SDS-PAGE.
Accessibility of ER Membrane Proteins to
Proteolytic Degradation
Consistent with its proposed role in ribosome binding, it
has been reported that bound ribosomes protect Sec61p
from digestion with exogenous proteases (Kalies et al.,
1994
Experiments were also performed to determine if bound
ribosomes afforded protease protection to the ribosome
receptors p180 and p34 (Savitz and Meyer, 1990 Relationship between Ribosome Structure and
Sec61p Accessibility
To further explore the correlation between membranebound ribosomes and the protease accessibility of Sec61p,
canine and porcine RM were treated with increasing concentrations of EDTA, and subsequently assayed for the
release of bound ribosomal subunits and Sec61p protease
accessibility. It has been previously demonstrated that exposure of RM to increasing concentrations of EDTA yields
preferential release of the small ribosomal subunit, and
partial release of the large subunit (Sabatini et al., 1966
Sec61p-associated Ribosomes Do Not Release Upon
Run-Off Translation
The data presented in Figs. 1 and 2 indicate that in native
RM, bound ribosomes protect Sec61p from digestion with
exogenous proteases and that release of bound ribosomes,
by addition of EDTA, is accompanied by a dramatic increase in protease sensitivity. In vivo, this behavior is likely
mimicked by the termination reaction, which yields the release of the nascent chain and the dissociation of the ribosome into its component subunits. To determine whether nascent chain termination on membrane-bound ribosomes
results in ribosome release and an increase in Sec61p protease accessibility, the accessibility of Sec61p to proteolytic
degradation was assessed following run-off translation. As
depicted in Fig. 3, incubation of RM with reticulocyte lysate,
either at 4°C (lanes 7-9) or at 25°C (lanes 10-12) did not
alter the protease susceptibility of Sec61p, an observation
consistent with a lack of ribosome release following runoff translation. Similar conclusions have been previously reported regarding rat liver RM (Sabatini and Blobel, 1970
80 S Ribosomes Fail to Compete with Ribosome/pPl 86 for Binding to RM
It has recently been reported that Sec61p displays nanomolar affinity for translationally inactive ribosomes at both
low and physiological salt concentrations and thus represents the predominant site of ribosome-membrane interaction in RM (Kalies et al., 1994
EKRM Containing Proteolyzed Sec61p Support
Ribosome/Nascent Chain Binding
Having acquired evidence suggesting that ribosome/nascent
chain binding to RM may involve multiple sites and/or mechanisms of association, we determined whether the binding
of ribosome nascent chain complexes displayed a requirement for intact Sec61p. EKRM were treated with chymotrypsin, under conditions in which Sec61p is quantitatively
clipped, and, following reconstitution of the membranes
with the 52-kD fragment of the SRP receptor, used in
binding reactions with pPl 86. To assess the structural state
of various ER integral membrane proteins, chymotrypsintreated EKRM were immunoblotted for Sec61p, SR
RM and EKRM Binding Capacity Does Not Correlate
with Accessible Sec61p
If Sec61p is the primary site of ribosome/nascent chain association with the ER membrane, the binding capacity of
RM for targeted nascent chains should correlate with the
concentration of free, or accessible, Sec61p. For example,
from the protease protection data in Fig. 1 and Sec61p
quantitative immunoblots (data not shown), there appear
to be ~200 fmol of available Sec61p per equivalent of canine RM (20% accessible, 1,000 fmol/eq. total) (Kalies et al.,
1994
Characteristics of Ribosome/pPl 86 Binding
The results of the binding capacity experiments shown in
Fig. 6 indicated that at limiting RM concentrations (0.25 eq.), the molar quantity of targeted and bound ribosome/
pPl 86 exceeded that of protease-accessible Sec61p. To
further characterize the bound state observed under these
conditions, four components of the binding reaction were
examined; dependence on the SRP receptor (SR To determine whether the binding observed at saturating nascent chain/membrane ratios was dependent on SR
As noted, salt- and protease-resistant binding of the pPl
86 to RM is thought to reflect ribosome association with
Sec61p and the insertion of the nascent chain into the
translocation site, itself defined primarily by Sec61p (Connolly and Gilmore, 1986 As a means of further characterizing the binding observed at saturation, pPl 86 was synthesized in the presence of either 0.25 or 1 eq. RM, treated with either 0.15 or
0.5 M KOAc, and centrifuged to separate membrane associated and membrane extracted nascent chains. Surprisingly, and as shown in Fig. 7 C, lanes 1 and 2, although
>75% of the pPl 86 remains bound to 0.25 eq. RM following extraction with physiological salt, only 35% is membrane associated following extraction with 0.5 M KOAc
(Fig. 5 C, lanes 1 and 2, and 5 D). In this experiment, the
minor, faster migrating translation product observed in
lane 2 P represents signal processed pPl 56-mer which is
generated upon salt-dependent dissociation of the ribosomal subunits (Murphy III, E.C., and C.V. Nicchitta, unpublished observations). In contrast to these results, yet in complete agreement with previous studies, pPl 86 synthesized in the presence of 1.0 eq. of RM is completely resistant to extraction with 0.5 M KOAc (Fig. 7 C, lanes 3 and
4) (Connolly and Gilmore, 1986 pPl 86 Bound At Saturation Is a
Posttargeting Intermediate
The kinetics and regulation of the transfer of the nascent
chain from the SRP/SR The bound state, whether obtained at limiting, or nonlimiting RM concentrations, is notable for the remarkable
degree of protease resistance displayed by the nascent chain
(cf. Fig. 7 B). If, under limiting RM concentrations, the
ribosome/nascent chain complex is bound to the RM solely
through physical association with SR
Analysis of pPl 86/Sec61p Interactions
As an additional means of characterizing the binding observed at saturation, chemical cross-linking was employed
to assess the molecular environment of the nascent chain.
Equivalent amounts of pPl 86 were translated in the presence of 0.25 or 1.0 eq. of RM and chilled to 4°C. RM were
recovered by centrifugation, resuspended, and treated with
the hetero-bifunctional chemical cross-linker, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for time
periods ranging from 15 s-15 min. As depicted in Fig. 9,
MBS treatment of ribosome/pPl 86 RM complexes at 0.25 eq. (saturation) and 1.0 eq. resulted in the formation of a
43-kD cross-linked species, previously demonstrated to be
comprised predominantly of Sec61p (Nicchitta et al., 1995
In this communication, we report the identification of a
novel stage of ribosome/nascent chain binding to the ER
membrane. Although independent of the apparent ribosome occupancy state of Sec61p, the described binding event
yields a nascent chain which is protected from digestion
with exogenous proteases, an established characteristic of
translocation competent, membrane-bound ribosome/nascent chain complexes (Connolly and Gilmore, 1986 The primary objective of these studies was to characterize ribosome/nascent chain binding using translationally active, nascent chain bearing ribosomes and native rough microsomes, an approach distinct from previous ribosomes
binding studies (Borgese et al., 1974 Current models of the mechanism of protein translocation into the ER indicate that vectorial translocation is a
consequence of topological restriction. In these models, a
tight junction between the ribosomal nascent chain exit site
and components of the protein conducting channel functions to restrict transit of the nascent chain to the ER lumen (Walter and Johnson, 1994 We observed that in both canine and porcine RM,
Sec61p was largely protected from protease digestion by
bound ribosomes. Conversely, treatment of canine and porcine RM with EDTA and 0.5 M KOAc, conditions known
to dissociate ribosomes from the ER membrane, resulted
in a dramatic increase in the sensitivity of Sec61p to proteolytic digestion. However, comparison of the binding capacity of RM and EKRM for ribosome/nascent chain complexes indicated that the binding capacity did not correlate
with the ribosome occupancy state of Sec61p. This was
surprising, as we had expected that removal of bound ribosomes from Sec61p would increase the total ribosome/nascent chain binding capacity. These data could be reconciled, however, if ribosome/nascent chain binding were comprised of sequential Sec61p independent and Sec61p
dependent stages. Such a proposal would also relieve the
stoichiometric constraints imposed by the observations,
from recent imaging studies of the ribosome/Sec61p complex, that a single ribosome binds three Sec61p complexes
(Hanein et al., 1996 Because it has been reported that inactive ribosomes
bind to Sec61p with nanomolar affinity (Kalies et al., 1994 To further characterize this novel binding event, binding reactions were performed as a function of membrane
concentration and the characteristics of the bound nascent
chain analyzed by established criteria. As has been shown
in previous reports, bound ribosome/pPl 86 was highly
protease and salt resistant when binding was performed in
the presence of 1 eq. of RM (Connolly and Gilmore, 1986 Could this novel binding stage represent a continued interaction of the ribosome/nascent chain/SRP complex with
SR We have provided direct evidence for the existence of a
novel stage of ribosome association with the ER membrane. The identification of this stage has significant ramifications for proposed mechanisms of protein translocation. First, it provides a means by which the translocon can
distinguish between cytosolic, free ribosomes and ribosomes engaged in the synthesis of secretory or membrane protein substrates. The translocon can, in effect, select ribosomes which are membrane associated via this novel
stage, thereby excluding those ribosomes which are cytosolic. In the absence of such a mechanism, one would expect competition between free ribosomes and ribosomes
synthesizing signal-bearing nascent chains (Nicchitta, 1996; Do et al., 1996
; Rapoport et al., 1996
; Hanein
et al., 1996
). Furthermore, it is thought that during translocation, the ribosome forms a tight, continuous seal with
Sec61p and thereby provides a direct, physically protected
path for the nascent chain as it passes from the exit site in
the ribosome to the protein conducting channel (Görlich
et al., 1992
; Crowley et al., 1993
).
; Stirling et al., 1992
).
When purified from mammalian sources, Sec61p is recovered as a complex containing two low molecular weight
subunits,
and
(Görlich and Rapoport, 1993
). Various temperature sensitive alleles of SEC61 display, at the nonpermissive temperature, profound defects in the translocation of a broad spectrum of secretory and membrane protein precursors (Rothblatt et al., 1989
; Stirling et al., 1992
).
Both in sequence and topology, Sec61p bears limited homology to SecY, a bacterial protein which, in concert with
SecA, SecE, and SecG, directs protein translocation across
the inner membrane of E. coli (Brundage et al., 1990
; Görlich et al., 1992
; Stirling et al., 1992
). Sec61p has been shown
by both chemical and photocross-linking approaches to be
in close physical proximity to translocating secretory and
integral membrane precursors, data consistent with the proposal that Sec61p is the protein conducting channel (Thrift
et al., 1991
; Görlich et al., 1992
; High et al., 1993a
,b;
Mothes et al., 1994
; Nicchitta et al., 1995
; Do et al., 1996
).
Related cross-linking approaches have also demonstrated
that phospholipids are physically proximal to the hydrophobic core of the signal sequence, suggesting that the
lipid bilayer can be directly accessed from the translocation site (Martoglio et al., 1995
).
; Crowley et al.,
1993
; Walter and Johnson, 1994
; Rapoport et al., 1996
; Hanein et al., 1996
). Indeed, it is generally assumed that there
exist protein components resident to the rough ER which
impart an affinity for ribosomes, and thereby yield the
morphological distinction between rough and smooth ER
(Blobel and Dobberstein, 1975
; Kreibich et al., 1978
). Historically, experiments designed to identify candidate ribosome receptors in the ER membrane have employed purified, inactive ribsomes and ER membranes stripped of
bound ribosomes (Borgese et al., 1974
). In these studies,
ribosomes were reported to bind to microsomes in a high
affinity, saturable manner (Borgese et al., 1974
). In rat
liver microsomes, high affinity, saturable ribosome binding
is markedly salt-sensitive, and is negligible at physiological
salt concentrations (Borgese et al., 1974
).
). From these data, it
appears that ribosome-nascent chain complexes bind to
discrete sites, defined by specific receptor proteins, and
that both the nascent chain and the ribosome contribute to
the binding event. The combination of these two binding
components yields a ribosome/nascent chain complex
which is resistant to salt extraction and digestion by exogenous protease (Sabatini and Blobel, 1970
; Adelman et al.,
1973
; Connolly and Gilmore, 1986
). There also appear to
be components of the ribosome which function in the
binding event. Recent reports on the regulation of ribosome binding to the ER membrane describe a role for the nascent chain-associated protein complex (NAC)1 in regulating the membrane binding activity of active ribosomes (Lauring et al., 1995a
). In the absence of the signal recognition particle (SRP), NAC has been demonstrated to
function as a global inhibitor of ribosome binding (Lauring et al., 1995b
).
(Savitz and Meyer,
1990
; Tazawa et al., 1991
; Ichimura et al., 1992
; Savitz and
Meyer, 1993
; Kalies et al., 1994
; Wanker et al., 1995
). p180
was identified through analysis of the inhibition of ribosome binding by protein fragments derived from proteolyzed ER membranes (Savitz and Meyer, 1990
, 1993
). In
reconstitution assays, p180 imparted ribosome binding activity to proteoliposomes (Savitz and Meyer, 1990
, 1993
).
Furthermore, proteoliposomes reconstituted from detergent extracts of RM depleted of p180 by immuno-affinity
chromatography, exhibited markedly reduced ribosome
binding, as well as defects in translocation (Savitz and
Meyer, 1993
). In addition, expression of p180 in yeast induces ER proliferation and an apparent increase in the
number of membrane-associated ribosomes (Wanker et al.,
1995
). Other groups have reported, however, that ribosome binding activity could be ascribed to a p180 deficient
protein fraction and thus the functional contribution of
p180 to ribosome binding is considered controversial (Collins and Gilmore, 1991
; Nunnari et al., 1991
). p34 was identified as an abundant protein component of liver ER membranes which, upon reconstitution into liposomes, exhibited ribosome binding activity (Tazawa et al., 1991
; Ichimura et
al., 1992
). Antibodies directed against p34 have been
shown to block ribosome binding and to impair translocation (Tazawa et al., 1991
; Ichimura et al., 1992
). In a
recent study, however, it was reported that p34 was not
protected from proteolytic digestion by membrane-bound ribosomes and thus was unlikely to mediate membrane
binding of ribosomes (Kalies et al., 1994
).
). Release of Sec61p from the RAMP fraction was achieved following treatment with high salt concentrations (0.75-1 M KOAc) and puromycin, conditions similar
to those employed to release ribosomes from intact RM
(Adelman et al., 1973
; Görlich et al., 1992
). Furthermore,
velocity sedimentation studies of solubilized RM indicated
that Sec61p solubilized at moderate (0.5 M) salt concentrations remains in association with the 80 S ribosome (Görlich et al., 1992
). In more recent studies, Kalies et al. (1994)
have provided direct evidence that at physiological
salt concentrations, Sec61p is the predominant ribosome
binding site in ER membranes (Kalies et al., 1994
). Using
native membranes as well as proteoliposomes containing
the purified Sec61p complex, Kalies et al. (1994)
identified
high affinity ribosome binding to the Sec61p complex at
physiological salt concentrations (Kalies et al., 1994
). As
would be predicted from the binding data, in native RM
the majority of Sec61p was found to be protected from proteolytic degradation by native, bound ribosomes (Kalies
et al., 1994
). It appears, therefore, that Sec61 is the primary ribosome receptor, although other components, such
as p180 and p34 may contribute to the total ribosome
binding activity.
Materials and Methods
. 60 S and 40 S subunits were resolved
on 10-30% sucrose gradients, following puromycin/0.5 M KOAc treatment, and separated subunit fractions resolved on SDS-PAGE. Strips of
SDS-PAGE gels containing either homogenous proteins L3/L4 or protein
S9 were excised, minced, mixed in Freunds complete adjuvant, and used
for antibody production in chickens. Animal services were performed by
contract agreement with Cocalico Biologicals (Reamstown, PA).
) containing a
cDNA insert encoding for bovine preprolactin, was linearized within the
coding region with PvuII. Transcription reactions were performed by the
procedure of Weitzmann et al. (1990)
in a buffer containing 40 mM Tris/
HCl (pH 8.0), 8 mM Mg(OAc)2, 25 mM NaCl, 2 mM spermidine, 10 mM
dithiothreitol, 2.5 mM ATP, CTP, UTP, and GTP, 2 U/ml yeast inorganic
pyrophosphatase and 1 U/ml T7 RNA polymerase. Cell-free translations
were performed in a rabbit reticulocyte lysate system as described (Nicchitta and Blobel, 1989
). Translations (20 µl) contained 8 µl of nucleasetreated rabbit reticulocyte lysate, 16 µCi of [35S] Pro-Mix (methionine/cysteine), 0.05 U/ml RNasin, 1 mM DTT, and 20 µM (
) methionine amino
acid mix. Reactions were adjusted to 110 mM KOAc, 2.5 mM Mg(OAc)2.
Rabbit reticulocyte lysate was prepared by the method of Jackson and
Hunt (1983)
and canine pancreas rough microsomes (RM) prepared by
the method of Walter and Blobel (1983)
. Translations were performed for
30 min at 25°C.
80°C.
Activity (52 kD)
was performed as described in Nicchitta and Blobel (1989).
. 20 ml of RM, at a concentration of 1 eq./ml, were diluted
1:1 with a buffer consisting of 1 M KOAc, 10 mM EDTA. After a 30-min
incubation on ice, RM were chromatographed, with upward flow, on a
170-ml Sepharose CL-2B column in 500 mM KOAc, 5 mM EDTA, 10 mM
2-mercaptoethanol, at a flow rate of 12 ml/h. The ribosome-stripped RM
fractions were pooled and centrifuged for 1 h at 40 K in the Ti50.2 rotor.
To remove the lumenal contents, pelleted membranes were resuspended
in 0.1 M Na-CAPS, pH 10.5, 10 mM 2-mercaptoethanol, incubated on ice for
30 min, and recovered by centrifugation for 40 min at 45 K in the Ti50.2
rotor over a cushion of 0.5 M sucrose, 50 mM K-Hepes, pH 7.2 (Nicchitta
and Blobel, 1993
). The ribosome and lumenal protein depleted RM were resuspended in 15% glycerol, 750 mM NaCl, 25 mM K-Hepes (pH 7.2), 10 mM 2-mercaptoethanol (buffer A), and solubilized by addition of Nikkol
to 1.5%. A high speed supernatant, containing soluble Sec61p, was obtained by centrifugation of the detergent/membrane mixture for 1 h at 45 K
in the Ti50.2 rotor. The soluble fraction was subsequently depleted of glycoprotein components by chromatography on a 10-ml con A-Sepharose
column, equilibrated in buffer A supplemented with 0.25 mg/ml egg yolk
phosphatidylcholine (PC), at a flow rate of 1.5 ml/h. The flowthrough fraction, depleted of glycoproteins was then chromatographed on a Superdex
200/60 gel filtration column, equilibrated in buffer A adjusted to 500 mM
NaCl, 0.5% Nikkol, 0.1 mg/ml PC at a flow rate of 0.5 ml/min. The Sec61p
enriched fractions, identified by immunoblot with an NH2-terminal directed Sec61p antibody, were pooled and dialyzed overnight against buffer A adjusted to 50 mM NaCl, 0.25% Nikkol, and 0.1 mg/ml PC. After dialysis, the protein fraction was centrifuged for 30 min at 45 K in the Ti50.2 rotor, to remove aggregates, and the supernatant chromatographed on a
5-ml Q-Sepharose FF column, equilibrated in dialysis buffer. The flowthrough fractions were directly loaded onto a Mono S 10/10 column and
eluted with a gradient of 50-500 mM NaCl in 25 mM K-Hepes, pH 7.4, 0.25% Nikkol, 0.1 mg/ml PC, 10 mM 2-mercaptoethanol. Peak Sec61p
containing fractions were pooled and concentrated in a Centricon 30 ultrafiltration device. On the basis of Coomassie blue staining, Sec61p purifed by this protocol was ~60% pure. Quantitation of the Sec61p content
of pH 10.5 washed RM (Nicchitta and Blobel, 1993
) was performed by quantitative immunoblot using purified Sec61p as standard and by densitometric analysis of Coomassie blue-stained gels, also with purified Sec61p
as standard.
80°C. Ribosome concentrations were determined using the relationship 1A260 = 21.4 pmol 80S
ribosomes (Martin et al., 1969
)
Results
). This observation was confirmed in experiments depicted in Fig. 1 A. In these experiments, RM were treated
with increasing concentrations of chymotrypsin at 4°C, and
immunoblotted with a polyclonal antibody directed against
the NH2 terminus of Sec61p. In canine and porcine RM, Sec61p is insensitive to proteolytic degradation at chymotrypsin concentrations up to 200 µg/ml. In five independent experiments with native RM, canine Sec61p was
>80% protected, while porcine Sec61p was >95% protected. Proteolytic cleavage of the Sec61p was assayed as
the appearance of a limit digestion product, indicated by
the asterisk, which maintains reactivity with the polyclonal antibody. To determine the contribution of bound ribosomes to the observed protection, ribosomes were extracted from RM by treatment with 15 mM EDTA and 0.5 M
KOAc (EKRM). As depicted in Fig. 1 B, upon removal of
bound ribosomes, the sensitivity of Sec61p to proteolytic
digestion is dramatically enhanced, with degradation occurring at chymotrypsin concentration as low as 10 µg/ml.
At higher chymotrypsin concentrations, >95% of Sec61p
is degraded to the limit digestion product. These data suggest that in native RM, the vast majority of the Sec61p exists in association with bound ribosomes and by virtue of
this association, is protected from proteolytic degradation
by exogenous proteases.
Fig. 1.
Protease protection of ER proteins in RM
and EKRM. 4 eq. of either
RM (A and C) or EKRM (B)
were diluted to 20 µl in a buffer containing 25 mM K-Hepes,
pH 7.2, 25 mM KOAc, and
2.5 mM Mg(OAc)2 at 4°C.
Samples were treated with
chymotrypsin (CT) at the
indicated concentration for
30 min at 4°C and reactions
quenched by addition of
TCA to 10%. After centrifugation, samples were processed for SDS-PAGE and
immunoblotted with antibodies directed against Sec61p, p180, and p34, as described
in Materials and Methods.
The migration of full-length
Sec61p, p180, and p34 are indicated by arrows; a prominent limit digestion product
of Sec61p is indicated by an
asterisk.
[View Larger Version of this Image (53K GIF file)]
; Tazawa
et al., 1991
; Ichimura et al., 1992
; Savitz and Meyer, 1993
,
1994; Wanker et al., 1995
). The results of these studies are
depicted in Fig. 1 C. In contrast to Sec61p, both p180 and
p34 were sensitive to digestion by chymotrypsin in native
RM. These data corroborate those of Kalies et al. (1994)
and indicate that of the proposed ribosome receptors
p180, p34, and Sec61p, only Sec61p is protected from proteolytic degradation by native, bound ribosomes.
).
RM were incubated in the presence of EDTA, and either
subjected to proteolysis with chymotrypsin, or centrifuged
to separate membrane associated and free subunits. In the
absence of EDTA treatment, >90% of the canine and
porcine Sec61p was protected from proteolytic digestion
(Fig. 2 A, lanes 2 and 9). Exposure to increasing concentrations of EDTA yielded a dramatic increase in the susceptibility of Sec61p to proteolytic digestion, an effect that
was somewhat more pronounced in canine RM. An immunoblot analysis of the distribution of the large ribosomal
subunit proteins L3 and L4 and the small subunit protein
S9 is shown in Fig. 2 B. Samples were also immunoblotted with an antibody directed against the ER integral membrane protein TRAP
, to insure that centrifugation conditions yielded complete recovery of the microsomal
membranes. Consistent with previous studies, treatment
of RM with EDTA resulted in the preferential release of
the small subunit and partial release of the large subunit
(Fig. 2 B, lanes 2-6). From these data it is apparent that
the protease accessibility of Sec61p appears coincident
with the dissociation/denaturation of bound ribosomes.
Fig. 2.
EDTA treatment
of RM: effects on Sec61p susceptibility to protease digestion and release of bound 60 and 40 S ribosomal subunits. 4 eq. of either canine or porcine RM were diluted to 20 µl
in buffer containing 25 mM
K-Hepes pH 7.2, 25 mM
KOAc at 4°C. After dilution,
samples were treated with
EDTA at the indicated concentration for 15 min at 4°C,
and either treated with chymotrypsin (25 µg/ml) for 30 min at 4°C (A), or centrifuged to separate membraneassociated and free ribosomal subunits (B). (A) After
chymotrypsin treatment and
acid precipitation, samples
were processed for SDSPAGE and immunoblotted
with an antibody directed
against Sec61p. As in Fig. 1,
the migration of both full-
length Sec61p and the limit digestion product are indicated. (B) After EDTA
treatment, samples were diluted sevenfold in a physiological salts buffer, layered over a 0.5-M sucrose cushion,
and centrifuged to separate
bound from free ribosomal
subunits (6 min, 60,000 rpm,
TLA 100 rotor, 4°C). Pellet
(P) and supernatant (S) samples were processed for SDSPAGE, and immunoblotted
with antibodies directed
against the 40-S subunit protein S9, 60 S subunit proteins
L3 and L4 (L3L4), or
TRAP.
[View Larger Version of this Image (71K GIF file)]
),
where it has also been directly demonstrated that the large
subunits of bound ribosomes do not undergo significant
exchange with free large subunits (Borgese et al., 1973
).
When EKRM were treated under identical conditions,
Sec61p remained highly sensitive to proteolytic digestion,
indicating that the concentrations of ribosomes present in
the reticulocyte lysate are insufficient to yield reprotection of the accessible Sec61p (Fig. 3, lanes 13 and 14).
Fig. 3.
Run-off translation does not alter the accessibility of
Sec61p to proteolytic digestion. RM were treated with buffer (25 mM K-Hepes, pH 7.2, 110 mM KOAc, 2.5 mM Mg(OAc)2) at 4°C
or 25°C, or with reticulocyte translation mixture (see Materials
and Methods) at 4°C or 2°C. Subsequently, samples were treated
with the indicated concentration of chymotrypsin for 30 min at
4°C. Samples treated with buffer were processed for SDS-PAGE
as described previously. Samples treated with reticulocyte translation mixture were diluted sevenfold in physiological salt buffer,
overlaid onto a 0.5-M sucrose cushion, and centrifuged for 10 min
at 60,000 rpm in a TLA 100 rotor, as described in the legend to
Fig. 2. The pellet (RM) fraction was solubilized in SDS-PAGE
sample buffer and processed for SDS-PAGE. After transfer to
nitrocellulose, samples were immunoblotted with an antibody directed against Sec61p.
[View Larger Version of this Image (27K GIF file)]
). While Sec61p appears to
mediate the majority of ribosome binding (cf. Figs. 1-3), it
has not been established whether Sec61p represents the
sole site of ribosome-membrane association, particularly
with regard to active, nascent chain bearing ribosomes.
Thus, a competition study was performed to determine if
free, inactive ribosomes compete with targeted, ribosome/
nascent chain complexes for binding sites on RM. In these
experiments, 80 S ribosomes were purified from reticulocyte lysate, and used in competition binding studies with pPl 86, a truncated, ribosome-associated form of preprolactin that is fully active in posttranslational targeting assays (Connolly and Gilmore, 1986
; Nicchitta and Blobel,
1989
). Increasing amounts of purified 80 S ribosomes were
added to RM, before the addition of ribosome/pPl 86 complexes and, following a 10-min incubation at 25°C, RM were
separated from the translation mix by centrifugation. Samples were resolved by SDS-PAGE and the relative amount
of bound and free nascent chains determined by phosphorimager analyses of the dried gels. All samples were normalized to a control sample lacking added ribosomes. In
the experiment shown in Fig. 4, 50 fmol of ribosome/pPl 86 substrate were incubated with RM in the presence of increasing concentrations of free, inactive ribosomes (0-19,000
fmol). Clearly, purified 80 S ribosomes compete quite poorly
with ribosome/pPl 86 complexes for membrane binding,
with a 25% inhibition observed at a 160-fold excess of free
ribosomes (Fig. 4). If there exist ribosome binding sites for
active, nascent chain bearing ribosomes, however, it should
be possible to compete radiolabeled ribosome/pPl 86 binding with an unlabeled ribosome/pPl 86 substrate. pPl 86 mRNA was thus translated in the presence and absence of
radiolabeled methionine, and membrane binding of the labeled substrate assayed in the presence of increasing
amounts of the unlabeled substrate. As shown in Fig. 4, unlabeled ribosome/pPl 86 was highly effective at competing
for radiolabeled ribosome/pPl 86 binding. Addition of one
translation equivalent of unlabeled substrate reduced
binding of labeled substrate by ~35% whereas a 1.5-fold
excess of unlabeled precursor reduced binding by almost 50%. These results demonstrate that binding of ribosome/
pPl 86 complexes is saturable and specific and further suggest that there may exist, in addition to the previously
identified Sec61p defined binding sites, additional ribosome binding sites in the ER membrane, possibly specific
for nascent chain bearing ribosomes.
Fig. 4.
Inactive ribososomes do not compete for binding with
ribosome/nascent chain complexes. pPl 86 was translated in the
absence of RM, and aliquots of the translation reaction (20 µl)
added to 1 eq. of RM, subsequent to addition of the indicated
quantity of 80 S reticulocyte lysate-derived ribosomes. Buffer conditions were adjusted such that the final KOAc concentration
was 140 mM. After incubation at 25°C for 10 min, RM-bound pPl
86 was separated from unbound by centrifugation through a 0.5-M
sucrose cushion as described in the legend to Fig. 2, and samples
processed for SDS-PAGE. Quantitation of the [35S] pPl 86 was
performed by phosphorimager analysis of the dried gels on a Fuji
MacBAS 1000 phosphorimager.
[View Larger Version of this Image (21K GIF file)]
,
TRAM, and the 22/23-kD subunit of the signal peptidase
complex (Fig. 5 A). From these data, it is apparent that under these conditions, Sec61p and SR
are fully degraded
whereas TRAM is >85% proteolyzed. Signal peptidase (SPC) is a stable heterooligomeric complex and data with
the SPC 22/23-kD subunit were included as a control for
excessive proteolysis. As noted, digestion of EKRM under
conditions which degrade Sec61p, also results in digestion
of SR
. Therefore, in order to restore physiological targeting, the proteolyzed membranes were reconstituted with
the cytoplasmic domain of SR
(Gilmore et al., 1982a
,b;
Meyer et al., 1982
). The activity of the reconstituted membranes in the targeting assay is shown in Fig. 5 B. In the
absence of RM, or in the presence of proteolyzed EKRM,
>90% of the pPl-86 is recovered in the supernatant fraction (Fig. 5 B, lanes 1 and 2). After reconstitution with the
SR
receptor fragment, ribosome/pPl 86 binding was restored (Fig. 5 B, lane 3). We conclude from these data that
intact Sec61p, and perhaps TRAM, are not required for the initial binding of active ribosome/nascent chain complexes to the ER membrane.
Fig. 5.
Proteolysis of Sec61p does not alter ribosome/pPl 86 binding. (A) Untreated and chymotrypsin-treated EKRM were
resolved on 12.5% SDS-PAGE gels, and immunoblotted with antibodies directed against Sec61p, SR, TRAM, and the 22/23-kD
subunit of the signal peptidase complex. (B) EKRM were treated
with chymotrypsin at 25 µg/ml for 30 min at 4°C, conditions known
to yield nearly quantitative digestion of Sec61p. After protease
treatment, chymotrypsin-treated EKRM were tested for their ability to support binding of ribosome/pPl 86, either in the presence
or absence of the 52-kD fragment of SR
. pPl 86 was translated
in the absence of 1.0 eq. EKRM (lane 1), in the presence of 1.0 eq. chymotrypsin-treated EKRM (lane 2), and in the presence of
1.0 eq. chymotrypsin-treated EKRM supplemented with 52 kD
SR
fragment (lane 3). The 52-kD SR
fragment was incubated with chymotrypsin-treated EKRM for 30 min at 4°C, before
translation.
[View Larger Version of this Image (45K GIF file)]
), and ~60 fmol of Sec61p per equivalent in porcine
RM (5% accessible, 1,200 fmol/eq.). Also depicted in Fig.
1 is the observation that virtually 100% of Sec61p is protease accessible in both canine and porcine EKRM, suggesting (at a minimum stoichiometry of 1 ribosome:
Sec61p complex) a binding capacity of ~1,000 fmol of nascent chains/RM equivalent for EKRM. An experiment testing this hypothesis is depicted in Fig. 6 A. From determinations of the specific activity of the [35S]methionine pool
and the incorporation of radiolabel into nascent chains,
the molar concentration of newly synthesized nascent
chains was calculated and, following isolation of the RM
by centrifugation, the quantity of bound vs free ribosome/
nascent chain complexes determined. In Fig. 6 A, ~150
fmol of ribosome/pPl 86 complex were incubated with
quantities of RM or EKRM ranging from 0 eq. (lane 1) to
1 eq. (lane 6). For each condition depicted, and at the levels of pPl 86 translation in the reticulocyte lysate system, maximal binding (>75% bound) was seen using 0.25 eq./
reaction (lane 4), operationally defined as saturated binding. No further increase in binding was observed when 0.5 eq. (lane 5) or 1 eq. (lane 6) were used. The data are depicted graphically in Fig. 6 B. These results are evident
from two significant points: First, and unexpectedly, EKRM,
in which Sec61p is virtually 100% protease accessible, did
not display an increased binding capacity for active ribosome/nascent chain complexes, relative to RM. Second, in
the depicted experiment, 0.25 equivalents of RM bound
~100 fmol of ribosome/nascent complexes, when, as determined by protease accessibility, only 15 fmol of Sec61p,
were available. It does not appear that the binding capacity in excess of the available Sec61p can be attributed to
bound ribosomes dissociating from Sec61p upon runoff
translation, as the data in Fig. 3, as well as previous reports, indicate that this does not occur (Sabatini and Blobel, 1970
; Borgese et al., 1973
). Thus, the binding capacity of RM for targeted, ribosome-associated nascent chains is
apparently insensitive to the ribosome occupancy state of
Sec61p. These results suggest that either there are multiple
sites of ribosome/nascent chain binding to the ER membrane or that the binding reaction is comprised of multiple
stages, with ribosome/nascent chain-Sec61p association
representing a stage subsequent to an initial binding event.
Fig. 6.
Ribosome/pPl 86 binding capacity of RM and EKRM.
pPl 86 was translated in the absence of microsomes and, following translation, 20-µl aliquots of the translation were added to the
indicated quantities of either RM or EKRM and a binding reaction performed for 10 min at 25°C. Samples were diluted sevenfold
using a buffer containing 25 mM K-Hepes, pH 7.2, 110 mM KOAc,
and 2.5 mM Mg(OAc)2, and overlayed onto a 0.5 M sucrose cushion. RM were collected by centrifugation (6 min, 60,000 rpm, TLA
100 rotor, 4°C). Supernatants were fractionated by ammonium
sulfate precipitation and prepared for SDS-PAGE as described
in Materials and Methods. [35S] pPl 86 was quantitated using a
Fuji MacBAS1000 phosphorimaging system. A digital image of the
dried gels is depicted in A, and the data depicted graphically in B.
[View Larger Version of this Image (39K GIF file)]
) activity, protease accessibility of the nascent chain, salt sensitivity of ribosome/pPl 86 binding, and nascent chain proximity to Sec61p. It is well established that under standard
assay conditions, membrane-associated pPl 86 is highly resistant to proteolytic digestion and remains associated with
the RM following extraction with high salt (Connolly and
Gilmore, 1986
; Nicchitta and Blobel, 1989
; Jungnickel and
Rapoport, 1995
). Conversely, binding of shorter ribosome/
pPl truncations (51-64 residues) has been shown to be salt
and protease sensitive (Jungnickel and Rapoport, 1995
).
,
and thus represented physiological targeting, RM were first
treated with N-ethylmaleimide (NEM) to inactive SR
(Gilmore et al., 1982b
; Nicchitta and Blobel, 1989
). After NEM
treatment, RM were tested for their ability to support
ribosome/nascent chain targeting and binding. As depicted in Fig. 7 A, in the presence of 1 eq. of RM, >80% of the
pPl 86 is recovered with the RM in the pellet fraction (lane 2)
whereas <15% of the pPl 86 is recovered in the membrane
fraction when translation is performed in the presence of
NEM-treated membranes (lane 3). Addition of the cytoplasmic domain of SR
to NEM-treated RM before targeting yielded reconstitution of binding activity (Fig. 7 A,
lane 4). These data indicate that the pPl 86 binding observed in the reticulocyte lysate/canine RM system is strictly dependent upon the activity of SR
and is therefore specific.
Fig. 7.
Characteristics of ribosome/pPl 86 binding. (A) Dependence on SR activity. pPl 86 translations were performed in the
absence of RM (lane 1) or the presence of RM (lane 2). An identical translation was done in the presence of NEM-treated RM
(lanes 3 and 4) (see Materials and Methods). In lane 4, the NEMtreated membranes were reconstituted with the 52-kD fragment of
SR
before translation. After translation, samples were diluted
sevenfold, layered over 0.5 M sucrose, and processes by centrifugation, as described in the legend to Fig. 3. Pellet and supernatant
samples were processed as previously described and resolved on
SDS-PAGE gels. (B) Dependence on SR
activity at 0.25 eq.
RM. pPl 86 translations were performed in the presence of 0.25 eq. RM (lane 1), or NEM-treated RM (lane 2). In lanes 3-7, pPl
86 was translated either in the absence of RM (lanes 3 and 4), or
in the presence of 0.25 (lane 5), 0.5 (lane 6) or 1.0 eq. of RM (lane
7). Subsequent to translation, samples were either left untreated
(lane 3) or digested with proteinase K (100 µg/ml) for 30 min at
4°C (lanes 4-7). Samples were resolved by SDS-PAGE (C) Saltextraction of bound translation products. pPl 86 was translated in
the presence of 0.25 (lanes 1 and 2) or 1.0 eq. RM (lanes 3 and 4).
After translation, samples 2 and 4 were diluted sevenfold in buffer
yielding a final concentration of 0.5 M KOAc. After a 15-min incubation at 4°C, the reactions were fractionated by centrifugation, as described in the legend to Fig. 3, and pellet and supernatant samples processed for SDS-PAGE. Quantitation was
performed by phosphorimager analysis; all translation products
were included in the analyses. (D) Graph of data described in C,
with the inclusion of samples containing 0.5 and 0.75 eq. RM. The
percent bound at physiological salt (150 mM KOAc) has been normalized to 100%.
[View Larger Versions of these Images (48 + 35K GIF file)]
; Rapoport et al., 1996
). Given the
suggestion, derived from the binding studies depicted in
Fig. 6, that Sec61p was not mediating the entirety of the
pPl 86 binding occurring at 0.25 eq. of RM, the binding
characteristics of the reaction performed under these conditions were determined. In the experiment shown in Fig.
7 B, lanes 3-7, pPl 86 was synthesized either in the absence
of RM (lanes 3 and 4), or in the presence of 0.25 (lane 5),
0.5 (lane 6), or 1.0 eq. (lane 7) of RM. The efficiency of the
binding reaction was then assayed by protease accessibility. In the absence of RM, virtually 100% of pPl 86 is sensitive to proteinase K (Fig. 7 B, lanes 3 and 4). The lower
molecular weight, limit digestion product seen in lane 4 represents that portion of the pPl 86 which resides within the
ribosome, and is thus inaccessible to protease (Malkin and
Rich, 1967
; Connolly and Gilmore, 1986
; Nicchitta and
Blobel, 1989
). When translation was performed in the
presence of 0.25 equivalents of RM, >75% of the pPl 86 was resistant to proteolytic digestion (Fig. 7 B, lane 5).
This is equivalent to that fraction recovered in association
with the membrane by sedimentation (Fig. 7 B, lane 2).
Similarly, when translations were performed in the presence of 0.5 or 1.0 equivalent of RM, ~90% of pPl 86 was
resistant to protease digestion (Fig. 7 B, lanes 6 and 7), corresponding once again to the bound fraction in the sedimentation assay. Note that the binding reactions performed at 0.25 eq. were, as expected, dependent upon
functional SR
(Fig. 7 B, lanes 1 and 2). From these data,
it is clear that at both saturating (0.25 eq.) and nonsaturating conditions (0.5 eq. and 1.0 eq.), RM bound pPl 86 nascent chains are resistant to proteinase K digestion, a characteristic of membrane inserted nascent chains (Connolly and Gilmore, 1986
; Nicchitta and Blobel, 1989
; Jungnickel
and Rapoport, 1995
).
; Nicchitta and Blobel, 1989
;
Jungnickel and Rapoport, 1995
). Thus, when translations
are performed under conditions in which the membrane
association reaction(s) is/are saturated, the majority of the
nascent chains are bound to the membrane, as assayed by protease accessibility, yet are sensitive to extraction with
0.5 M KOAc. These observations define a novel state of ribosome/nascent chain association with the ER membrane.
bound state to Sec61p have not
been elucidated. In the absence of this information, it must
be considered that at binding saturation, a substantial fraction of the bound ribosome/nascent chain complexes may
exist in a stable complex with SRP and SR
, and thus comprise targeting, rather than membrane-bound intermediates. To test this hypothesis, translation reactions were performed at saturating ribosome/nascent chain ratios and
the interactions between the ribosome/nascent chain complex and SR
investigated.
, it would be predicted that proteolytic degradation of SR
would result in
release of the ribosome/nascent chain complex from the membrane. The experiment depicted in Fig. 8 was performed to test this prediction. pPl 86 was translated in the
presence and absence of limiting RM concentrations and,
consistent with previous data, was recovered in the pellet
fraction when translations were performed in the the presence, but not the absence, of RM (Fig. 8, lanes 1 and 2).
Also consistent with previous observations, membrane associated, but not free, pPl 86 was protected from digestion with exogenous proteases (Fig. 8, lanes 3 and 4). Significantly, the entire population of membrane bound nascent
chains remained associated with the membrane following
protease digestion (Fig. 8, lane 5). Under these conditions,
SR
was completely degraded (Fig. 8, lanes 7 and 8). Thus,
when pPl 86 is bound to RM at limiting RM concentrations, SR
is fully accessible to proteolytic digestion, yet the
association of the ribosome/nascent chain complexes with the membrane is unaltered. These data indicate that the
bound form of the pPl 86 obtained at limiting RM concentrations is, in fact, a posttargeting intermediate and does
not arise through stable association with SR
.
Fig. 8.
Analysis of ribosome/nascent chain-SR complex formation. pPl 86 was translated in the presence or absence of 0.25 eq. of RM. Aliquots of the translation were removed and the
membrane-bound translation products resolved by sedimentation
analysis (lanes 1and 2), or subjected to proteolysis with 100 µg/ml
proteinase K for 30 min at 4°C, before sedimentation (lanes 3-5).
Fractions were processed as described in the legend to Fig. 3.
Paired samples were processed in parallel for immunoblot analysis of SR
.
[View Larger Version of this Image (28K GIF file)]
).
This complex was seen at all time points assayed, at both
saturating (Fig. 9 A) and nonsaturating (Fig. 9 B) conditions, and reached a maximum within the time frame of the
experiment (Fig. 9 C). Quantitation of the pPl 86/Sec61p
cross-linked complex formed under saturating and nonsaturating conditions revealed that twice as much pPl 86 became cross-linked to Sec61p under nonsaturating conditions (Fig. 8 C). These data suggest that when membrane
binding sites are limiting, the topological relationship between the population of bound ribosome/pPl 86 complexes and Sec61p is significantly altered from that when
membrane binding sites are in excess. At saturation, it
appears that a significant fraction of the bound ribosome/
pPl 86 complexes are not in the physical proximity of
Sec61p.
Fig. 9.
Cross-linking of bound pPl 86 to Sec61p at 0.25 eq. RM
and 1.0 eq. RM. pPl 86 was translated either in the presence of
0.25 (A) or 1.0 eq. (B) of RM and processed for cross-linking with the heterobifunctional cross-linker MBS, as detailed in Materials and Methods. Cross-linking reaction times ranged from 15 s-15 min, and reactions were quenched by addition of 1 vol of PBS
supplemented with 50 mM DTT, 50 mM lysine, 1% SDS. Samples were TCA precipitated and resolved on 12.5% SDS-PAGE
gels. The positions of pPl 86 and the cross-linked pPl 86/Sec61p
are indicated. Quantitation was by phosphorimager analysis (Fuji
MacBAS 1000). (C) Graph of data derived from A and B.
[View Larger Versions of these Images (54 + 23K GIF file)]
Discussion
; Nicchitta and Blobel, 1989
; Jungnickel and Rapoport, 1995
).
The identification of this binding reaction was dependent
upon two experimental manipulations. One, and in contrast to most previous studies, ribosome binding was assayed as the functional association of targeted ribosome/
nascent chain complexes with the ER membrane, rather
than binding of translationally inactive ribosomes (Borgese et al., 1974
; Savitz and Meyer, 1990
; Kalies et al., 1994
).
Secondly, binding reactions were performed as a function
of membrane concentration, which, under conditions where
membrane binding sites were limiting, revealed a novel,
protease resistant, salt-sensitive association of the nascent
chain with the ER membrane.
; Savitz and Meyer, 1990
;
Kalies et al., 1994
). Two experimental observations warranted this approach. Foremost, binding of nascent chainbearing ribosomes to RM is known to require a specific
targeting event, which limits ribosome binding to those ribosomes synthesizing signal or topogenic sequence bearing nascent chains. Furthermore, native RM, although
richly endowed with bound ribosomes, are fully functional
in vitro, and thus must contain binding sites relevant to the
translocation reaction. In addition to these experimental considerations, the observation that translocation in the
mammalian ER is strictly cotranslational indicates that the
mechanism and regulation of ribosome binding to the ER
membrane is intimately related to the mechanism and regulation of protein translocation (Blobel and Dobberstein,
1975
).
; Rapoport et al., 1996
). This
model is founded upon established experimental observations. For example, translocation-competent binding of truncated preprolactin precursors is accompanied by conversion of the nascent chain from a protease-sensitive, to a
protease-insensitive state, data interpreted to be indicative
of a tight junction (Connolly and Gilmore, 1986
; Nicchitta
and Blobel, 1989
; Jungnickel and Rapoport, 1995
). Subsequently, it was observed that the resident ER membrane
protein Sec61p, is highly enriched in the ribosome fraction
of solubilized ER membranes and is thus a likely candidate for the ribosome receptor (Görlich et al., 1992
). Sec61p has also reported to reside in close physical proximity to translocating secretory and membrane protein precursors (Thrift
et al., 1991
; Görlich et al., 1992
; High et al., 1993; Mothes
et al., 1994
; Do et al., 1995; Jungnickel and Rapoport,
1995
; Nicchitta et al., 1995
). In addition, Kalies et al. (1994)
observed high affinity binding of inactive ribosomes to
Sec61p and demonstrated that inactive ribosomes, in binding to Sec61p, protect it from proteolytic degradation
(Kalies et al., 1994
). Direct evidence for tight ribosomemembrane coupling was reported in studies of the quenching kinetics of nascent chains bearing fluorescent reporter
groups (Crowley et al., 1993
, 1994). In these studies, the
fluoresence of membrane-bound short nascent chains bearing reporter groups were demonstrated to be insensitive to
collisional quenching by exogenous iodide ions, indicating
that the ribosome-membrane junction is essentially contiguous. These data support a model in which the ribosome is tightly coupled to the ER membrane through direct physical interactions with Sec61p (Walter and Johnson, 1994
;
Rapoport et al., 1996
). While it is clear that binding of ribosome/nascent chain complexes culminates in a tight interaction with Sec61p, the mechanism by which this stage is
ultimately achieved is not. The answer to this question
bears significant ramifications on the mechanism and function of ribosome binding to the ER membrane.
).
),
we reasoned that if the described binding stage was independent of Sec61p, isolated ribosomes would not compete
for binding of targeted ribosome/nascent chain complexes.
In support of this hypothesis, addition of up to a 350-fold
molar excess of purified 80 S ribosomes failed to yield substantial competition for binding. In contrast, addition of a
1.5-fold molar excess of unlabeled ribosome/pPl 86 resulted in efficient binding competition. These results lend
credence to a model in which the initial binding event can occur independent of interactions with Sec61p. Most significantly, and in context of current models of the mechanism
of protein translocation, these data suggest that formation
of a protease-resistant ribosome-membrane junction can
precede physical interaction of the ribosome with Sec61p.
We have been as yet unable to identify a role for the ribosome receptors p180 and p34 in the binding of active, ribosome/nascent chain complexes. However, as both p180 and
p34 were identified in binding studies using inactive ribosomes, it is reasonable to speculate that the binding of active and inactive ribosomes are independently regulated.
In support of this, we have observed that under conditions
in which ribosome/nascent chain binding is saturated, there
is no apparent decrease in the relative protease sensitivity
of p180 or p34 (Murphy, E.C., and C.V. Nicchitta, unpublished observations).
;
Nicchitta and Blobel, 1989
; Jungnickel et al., 1995). However, when the same criteria were applied to samples containing 0.25 eq. RM, it was found that, while protease resistant, the majority of bound ribosome/pPl 86 was salt
extractable. That the binding obtained at 0.25 eq. is dependent upon physiological targeting is supported by the observation that bound nascent chains are protease resistant,
and that binding is blocked by treatment of the RM with
NEM, which, at the concentrations used, is known to inactivate the SRP receptor
subunit (Gilmore et al., 1982;
Nicchitta and Blobel, 1989
).
? All available evidence indicates that the interaction
of SR
with nascent chain-bound SRP results in SRP release (Gilmore et al., 1982a
,b; Miller et al., 1993
; Bacher et
al., 1996
). Furthermore, it is not clear how such a continued interaction would yield a protease resistant form of
the precursor, given that in the absence of RM, the nascent chain, with bound SRP, is readily digested by exogenous proteases (Connolly and Gilmore, 1986
; Nicchitta
and Blobel, 1989
; Jungnickel and Rapoport, 1995
; Fig. 5).
In addition, we observed that at saturation, SR
can be
readily and fully degraded by exogenous proteases, yet the
ribosome/nascent chain complexes remain bound to the
RM. Given these considerations, we favor a model in which the initial targeting reaction places the nascent chain in a
protease resistant, yet salt extractable, environment. We
postulate that at this stage, the ribosome is binding to factors other than Sec61p, and that the signal sequence is associated with components of the ER membrane through hydrophobic and electrostatic interactions, perhaps directly
involving the lipid bilayer. At a subsequent stage, occurring
coincident with the interaction of the ribosome with Sec61p,
the nascent chain is stably inserted into the translocon and
assumes a translocation competent conformation. This
model predicts an alternative binding site on the ER membrane for targeted ribosome-nascent chain complexes. The
identity of this site is currently under investigation.
).
Furthermore, we postulate that this novel stage provides the nascent chain the opportunity to attain a translocation
competent topology, perhaps through recruitment of translocon components in a manner first proposed in the signal
hypothesis (Blobel and Dobberstein, 1975
). In this scenario, only those precursor proteins that have properly assembled into the ER membrane are available for subsequent translocation by the translocon.
Please address all correspondence to C.V. Nicchitta, Duke University Medical Center, Department of Cell Biology, Durham, NC 27710. Tel.: (919) 684-8948. Fax: (919) 684-5481. E-Mail: chris_nicchita{at}cellbio.duke.edu
Received for publication 22 October 1996 and in revised form 31 January 1997.
We thank Drs. Edwin C. Murphy Jr., T. Bell, M. Likker, and S. Dias for critical comments, advice, and support through the course of these studies. We also wish to express our gratitude to Dr. David I. Meyer, for providing antibodies directed against p180 and helpful advice, and Dr. Stephen High, for providing antibodies to p34.
This work was supported by National Institutes of Health grant DK47897 (CVN).
EKRM, KOAc and EDTA washed RM; NAC, nascent chain-associated protein complex; NEM, N-ethyl maleimide; RM, rough microsome; SRP, signal recognition particle.