In Vitro Binding of Ribosomes to the
Subunit of
the Sec61p Protein Translocation Complex*
Robert
Levy
,
Martin
Wiedmann§, and
Gert
Kreibich
¶
From the
Department of Cell Biology, New York
University School of Medicine, New York, New York 10016 and
§ Cellular Biochemistry and Biophysics Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, June 6, 2000, and in revised form, September 15, 2000
 |
ABSTRACT |
The Sec61p complex forms the core element of the
protein translocation complex (translocon) in the rough endoplasmic
reticulum (rough ER) membrane. Translating or nontranslating ribosomes
bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present
evidence that the
subunit of the complex (Sec61
) makes contact
with nontranslating ribosomes. A fusion protein containing the Sec61
cytoplasmic domain (Sec61
c) prevents the binding
of ribosomes to stripped ER-derived membranes and also binds to
ribosomes directly with an affinity close to the affinity of ribosomes
for stripped ER-derived membranes. The ribosome binding activity of Sec61
c, like that of native ER membranes, is sensitive
to high salt concentrations and is not based on an unspecific
charge-dependent interaction of the relatively basic
Sec61
c domain with ribosomal RNA. Like stripped ER
membranes, the Sec61
c sequence binds to large ribosomal
subunits in preference over small subunits. Previous studies have shown
that Sec61
is inessential for ribosome binding and protein
translocation, but translocation is impaired by the absence of
Sec61
, and it has been proposed that Sec61
assists in the
insertion of nascent proteins into the translocation pore. Our results
suggest a physical interaction of the ribosome itself with Sec61
;
this may normally occur alongside interactions between the ribosome and
other elements of Sec61p, or it may represent one stage in a temporal
sequence of binding.
 |
INTRODUCTION |
The cotranslational translocation of proteins across the
endoplasmic reticulum (ER)1
membrane of eukaryotes involves the binding of translating ribosomes to
receptor sites on the ER membrane, where the nascent protein enters the
ER lumen through a proteinaceous pore. Translocation is preceded by a
targeting phase in which ribosomes bearing pre-secretory nascent chains
are selectively recruited to the ER membrane through the reciprocal
action of at least two cytosolic factors, the signal recognition
particle (SRP) (1) and the nascent polypeptide-associated complex (2).
An interaction between SRP and its membrane-bound receptor (3, 4) is
followed by transfer of the ribosome to the translocation site
(translocon) in the ER membrane, where the nascent peptide passes
directly from an aqueous tunnel or channel within the ribosome to an
aqueous pore within the membrane (5-8).
The core element of the translocon is formed by the Sec61p complex, a
heterotrimer of three transmembrane proteins (Sec61
,
, and
subunits) (9-14). Sec61
, the largest component of the complex,
contains 10 membrane-spanning domains (9, 15); the Sec61
and
components are both thought to be "tail-anchored" proteins, with a
predominantly cytoplasmic disposition (10, 16). Each functional complex
appears to contain 3 or 4 Sec61p heterotrimers (17).
Tranlocation has been reconstituted in vitro (18) with
membranes containing Sec61p in addition to only two other components as
follows: SRP receptor and the translocating chain associated membrane
protein (TRAM) (19), whose presence does not appear to be required for
the transport of all pre-secretory proteins (19, 20).
Photocross-linking experiments have shown that the nascent chain is
adjacent to Sec61
throughout its transit of the ER membrane (21).
Sec61p thus appears to form the hydrophilic translocation pore in the
ER membrane.
The ribosome-ER interaction depends on contacts between the body
of the ribosome and protein components of the ER membrane, in addition
to the interaction of the nascent chain and the translocation pore.
Treatment of rough microsomes (RM) with puromycin causes the expulsion
of nascent chains into the ER lumen (22), but the release of ribosomes
from the membrane requires additional exposure to high salt
concentrations (23); nontranslating ribosomes can rebind to
puromycin/high salt-stripped RM (PKRM) at physiological salt
concentrations (24). Nontranslating ribosomes also bind with high
affinity to artificial liposomes reconstituted with the Sec61p complex
alone (25). Therefore, Sec61p is believed to serve as both a
translocation pore and a ribosome-binding site. The isolated Sec61p
complex forms a roughly pentagonal doughnut-shaped structure (17, 26),
whose assembly in lipid membranes is induced by the presence of
ribosomes (17). The yeast Sec61p complex has been visualized in the
ribosome-bound state in detergent solution, where the central pore of
the complex appears to align with the presumed nascent chain exit site
on the large ribosomal subunit (26). In this reconstruction, the
connection between the ribosome and Sec61p is restricted to a thin
stalk, suggesting that other proteins may contribute to the tight
ER-ribosome seal in vivo or that the actively translocating
ribosome-ER complex assumes a different conformation.
The identity of the Sec61p components or domains involved in
ribosome binding remains open to speculation. Previous results suggest
that the
subunit is the least essential for the overall function of
the complex. Sec61
can be proteolytically degraded in rough
microsomes without release of bound ribosomes, whereas Sec61
remains
protected (25), and limited proteolysis of stripped RM has led to the
identification of Sec61
domains that may be required for ribosome
binding and translocation (48). Although yeast mutants lacking either
the
or the
subunit are inviable (11-13), a deletion of both
yeast Sec61
homologues (Sbh1p and Sbh2p) is not lethal and causes a
reduction in growth rate only at elevated temperatures (27). Disruption
of Sec61
in Drosophila, however, is lethal and causes
embryonic defects consistent with impaired secretion (28).
Sec61
-depleted proteoliposomes bind ribosomes with an affinity
comparable to that of proteoliposomes containing intact Sec61p (29).
Sec61
-depleted proteoliposomes showed impaired translocation
in vitro, but the impairment could be overcome by allowing
the SRP-dependent targeting step to occur at reduced
temperature, in the absence of ongoing translation. It was proposed
that Sec61
affects the kinetics of nascent chain insertion and that
in the absence of Sec61
insertion proceeds at a reduced rate,
allowing the competing process (elongation in the cytosol) to take
precedence. Proteolytic digestion of Sec61
in stripped rough
microsomes did not abolish their ability to translocate preprolactin
in vitro (30).
The present study addressed the physical interaction between ER
membrane proteins and core components of the ribosome. We found that
the purified Sec61
cytoplasmic domain (Sec61
c)
competes for ribosome binding to stripped ER membranes;
Sec61
c also binds to ribosomes directly, with an
affinity comparable to the affinity of ribosomes for stripped ER
membranes. Based on these observations we propose that Sec61
may
participate in ribosome binding and translocation by interacting
directly with the body of the ribosome. This interaction may occur
after, or concurrently with, an initial contact between the ribosome
and other domains of Sec61p, and it does not preclude an interaction
between Sec61
and the nascent chain.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction--
To construct pET28a-GST, the
GST-coding sequence of pGEX-EF, containing the Schistosoma
japonicum GST cDNA sequence, was amplified by PCR using
primers GST(+)/NcoI/SalI/Thr
(5'-CATGCCATGGGCGTCGACCTGGTGCCGCGCGGCAGCATGTCCCCTATACTAGGTTATTGG-3') and GST(
)/XhoI
(5'-CCGCTCGAGTTATTTTGGAGGATGGTCGCCACCACC-3'); the PCR product was
inserted into pET28a(+) (Novagen, Inc., Madison, WI) at
NcoI/XhoI sites. Primer
GST(+)/NcoI/SalI/Thr introduces a thrombin
cleavage site (Leu-Val-Pro-Arg-Gly-Ser) upstream of GST. The sequence
encoding the first 70 amino acids of Sec61
was amplified with
Sec61
(+)/NcoI
(5'-CATGCCATGGGCATGCCTGGTCCGACCCCCAGTTGGC-3') and Sec61
c
(
)/SalI (5'-ACGCGTCGACAGGGCCAACTTTGAGCCCAGGTGA-3'), and the PCR product was inserted into pET28a-GST at
NcoI/SalI sites to generate
pET28a-
c/GST. The Sec61
c sequence in the
resulting clone was confirmed by sequencing of both DNA strands.
Growth and Purification of GST Fusion Proteins--
Plasmids
pET28a-GST and pET28a-GST/Sec61
c were used to transform
Escherichia coli strain BL-21(DE3), and the corresponding fusion proteins were expressed by induction of the liquid cultures with
1 mM isopropylthio-
-D-galactoside for 1 h at 37 °C. The fusion protein was purified using
glutathione-agarose beads (Sigma) according to standard procedures (31)
and stored at 4 °C for up to 1 month. To elute GST fusion proteins,
a volume of beads was transferred to a disposable column (Bio-Rad) and
eluted with 5 mM glutathione, 50 mM Tris-HCl,
pH 8.0. The protein concentration of each fraction was estimated by
Bradford assay (32), using GST alone as a standard and using the value
1 A280 = 0.5 mg/ml to obtain the concentration
of GST. Eluted fusion proteins were stored at
70 °C.
Preparation of Ribosomes, Stripped Rough Microsomes, and
Ribosomal Subunits--
Canine pancreatic RM were prepared as
described (33). RM were stripped of ribosomes using puromycin/KCl (9),
and the ribosomal and membranous components were collected separately and used in subsequent experiments. To prepare large and small ribosomal subunits, RM were incubated with puromycin/KCl as described (9), and 2-ml aliquots of the suspension were layered on continuous density gradients (36 ml of total) of 7-20% sucrose in high salt buffer (HSB: 50 mM HEPES-KOH, pH 7.5, 0.5 M
KCl, 10 mM MgCl2) in Beckman SW-28 tubes. The
samples were centrifuged for 9 h, 25,000 rpm, 4 °C, in the
SW-28 rotor. 2-ml fractions were collected manually from the top of the
gradient. The RNA content of each fraction was determined by
A260 measurement. The pooled small and large
subunit fractions were each diluted with 1.5 volumes of HKM (50 mM HEPES-KOH, pH 7.5, 50 mM KOAc, 5 mM Mg(OAc)2), and 10-ml volumes of the sample
were layered over 0.5 ml of 1 M sucrose in ribosome binding
buffer (RBB: 50 mM HEPES-KOH, pH 7.5, 0.1 M
KOAc, 5 mM Mg(OAc)2, 2 mM
dithiothreitol) and 50 µl of 1.9 M sucrose/RBB in a
Beckman SW-41 tube. Each tube was topped off with HKM, and the samples
were centrifuged for 20 h, 34,000 rpm, 4 °C in the SW-41 rotor.
Pellets were resuspended in 0.25 M sucrose/RBB and stored
at
70 °C. Both the small subunit and the large subunit
preparations had
A260:A280 ratios of 2.0. The purity of each preparation was assessed by Western blotting using
antibodies against the ribosomal proteins S3 and L5. The small subunit
preparation was virtually free of large subunits, whereas the large
subunit preparation was slightly contaminated (20% or less) with small subunits.
Ribosomes were surface-labeled with
t-butoxycarbonyl-L-[35S]methionine
N-hydroxysuccinimidyl ester (35SLR; Amersham
Pharmacia Biotech) as described previously (25). The labeled ribosomes
were stored in 0.25 M sucrose/RBB and added directly to
binding experiments. The specific activity of the ribosome preparation
was typically about 5 × 106 cpm per mg of ribosomes
(34).
Assays for Ribosome Binding--
Binding of ribosomes to
puromycin/KCl-stripped rough microsomes (PKRM) was measured according
to an established method (24, 35). Briefly, labeled ribosomes were
mixed with 5-10 eq (33) of PKRM in 20 µl of RBB. After 30 min on
ice, the sample was mixed with 180 µl of 2.3 M
sucrose/RBB and overlaid with 360 µl of 1.9 M sucrose/RBB
in a 5 × 41-mm open top centrifuge tube (Beckman Instruments,
Palo Alto, CA). The samples were topped off with RBB and centrifuged
(75 min, 40,000 rpm, 4 °C, Beckman SW-50.1 rotor). The tubes were
frozen in liquid nitrogen and sectioned at points 1.5 and 2.5 cm from
the top of the tube. The ribosome content of the resulting top, middle,
and bottom fractions was determined by scintillation counting. The top
and bottom fractions were counted as bound and unbound, respectively,
whereas the middle fraction, containing negligible radioactivity, was
not included in subsequent calculations.
To test the effect of GST or Sec61
c/GST as competitors
in the ribosome binding assay, the purified protein (after elution from
glutathione-agarose; see above) was added to the binding assay in the
amounts indicated in the figure legends. The fusion protein was mixed
with labeled ribosomes before the addition of membranes. Compensating
volumes of elution buffer (5 mM glutathione/50 mM Tris-HCl, pH 8.0) were added so that each 20-µl
reaction contained 2.5 µl of fusion protein and/or elution buffer.
The presence of elution buffer alone in this amount did not affect
ribosome binding.
Direct binding of ribosomes to GST or Sec61
c/GST
immobilized on glutathione-agarose beads was measured as follows. 20 µl of beads with bound fusion protein were equilibrated in RBB and mixed with SLR-labeled ribosomes in a final volume of 0.1 ml. In some
experiments additional agents were added as described in the figure
legends. Each reaction was incubated on ice for 10 min with
intermittent agitation to resuspend the beads; the samples were then
incubated for 10 min on a rotating wheel at 4 °C and stored on ice
again for 10 min with intermittent agitation as before. 0.4 ml of RBB
was added to each sample to improve the separation of bound and unbound
material in the following step. The tubes were mixed once by inversion
and immediately spun for 30 s in a microcentrifuge at room
temperature. The supernatant was transferred to a new tube, and
residual liquid was removed with a Hamilton syringe. Equivalent volumes
of the bead fraction and the supernatant were measured by scintillation
counting. For calculations, 0.1 ml was taken as the reaction volume.
(In control experiments, addition of 0.4 ml of buffer at the end of the
initial binding reaction caused a relatively slow re-equilibration of ribosomes that did not affect the calculated values substantially.)
 |
RESULTS |
The results presented here developed out of a screen for the
ability of potential ribosome receptor domains to interfere with the
in vitro binding of ribosomes to stripped rough microsomes when expressed as glutathione S-transferase fusion proteins.
The Sec61
cytoplasmic domain ((Sec61
c) was placed
upstream of GST, with an intervening thrombin cleavage site (see
"Experimental Procedures"). In the resulting fusion protein, the
C-terminus of Sec61
c is fused to GST, whereas the N
terminus of Sec61
c is free; this corresponds to the
predicted disposition of the native Sec61
, whose C terminus is
thought to be anchored in the ER membrane (10). The fusion protein was
expressed in bacteria and purified using glutathione-agarose beads as
described under "Experimental Procedures." A construct encoding GST
without the added Sec61
c sequence was used in parallel
as a negative control. Fig. 1A
shows the purified GST and Sec61
c/GST with or without thrombin protease cleavage. The intact Sec61
c/GST fusion
protein without thrombin cleavage was used in all subsequent
experiments presented here. After elution from glutathione-agarose, the
GST and Sec61
c/GST fusion proteins, respectively, were
mixed with labeled nontranslating ribosomes (~100-fold molar excess
of fusion protein over ribosomes) before addition of stripped rough
microsomes. Ribosomes were chemically surface-labeled with sulfur
labeling reagent (SLR); this did not measurably affect the
membrane-binding properties of the ribosomes. The ribosome binding
assay was carried out as described under "Experimental Procedures."
The presence of GST alone in the reaction did not affect the extent of
ribosome binding appreciably, compared with a sample in which no
competitor was present (not shown). By contrast, the fraction of
membrane-bound ribosomes in the Sec61
c/GST-containing
sample was reduced to ~50% of the value obtained when GST alone was
added as a competitor (Fig. 1B). To get an estimate of the
amount of the fusion protein required to inhibit binding, we performed
competition assays with increasing amounts of
Sec61
c/GST. As shown in Fig. 1C, the
inhibition of binding did not follow a linear relation to the amount of
fusion protein added; up to a 16-fold excess of
Sec61
c/GST over binding sites (25-fold excess over
ribosomes) resulted in only a minor reduction in binding, whereas a
64-fold excess over binding sites caused a more substantial reduction
(fraction bound = 52% of the control value). GST normally
dimerizes in solution (36), and under the conditions used in our
ribosome binding experiments a significant portion of free GST formed
aggregates large enough to be recovered by centrifugation at
100,000 × g for 1 h (not shown). This behavior
may account partially for the relatively large excess of free
Sec61
c/GST needed to affect the binding of ribosomes to
stripped rough microsomes.

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Fig. 1.
A soluble GST fusion protein containing the
Sec61 cytoplasmic domain competes for
attachment of ribosomes to stripped rough microsomes.
A, expression of Sec61 c/GST. The GST and
Sec61 c/GST fusion proteins were expressed in bacteria
and purified using glutathione-agarose as described under
"Experimental Procedures." After elution with free glutathione, the
fusion protein was left untreated or else subjected to thrombin
digestion to cleave between the Sec61 c and GST sequences
(50 µg of fusion protein were incubated with 1 µg of thrombin
protease overnight at room temperature). To cleave between the Sec61
and GST sequences, equal aliquots of each sample (~5 µg per lane),
with or without prior thrombin treatment as indicated, were subjected
to SDS-PAGE on a 12% acrylamide gel, followed by Coomassie Blue
staining. The free Sec61 c sequence (~8 kDa) was
detected transiently in control experiments but was degraded by the
extended thrombin treatment needed for complete cleavage of the fusion
protein and is not seen in the figure. The uncleaved forms of both GST
and Sec61 c/GST were used in all subsequent experiments.
B, activity of 1 Sec61 c/GST in the ribosome
binding/competition assay using labeled nontranslating ribosomes. The
ribosome binding/competition assay was done as described in the text,
using an input of 2 µg of SLR-labeled ribosomes and 2 µg of the
indicated fusion protein. The excess of fusion proteins over ribosomes
was ~140:1, assuming a Mr of 4.5 × 106 for the ribosome and 3.2 × 104 for
each fusion protein. After 10 min on ice, 10 eq of PKRM was added, and
the reaction was incubated for an additional 30 min on ice, followed by
flotation to separate the bound and unbound fractions and scintillation
counting. The fraction bound (y axis) was calculated by
dividing the bound value by the total (bound plus unbound); this value
was set equal to 1 in the case of the control sample (GST alone), and
the fraction bound in the presence of Sec61 c/GST was
expressed relative to the control value. C, titration of the
Sec61 c/GST competitive activity. The binding experiment
was performed as in B. 2 µg of SLR-labeled ribosomes were
mixed with the indicated molar excess of the Sec61 c/GST
fusion over binding sites. (The number of binding sites was calculated
from the X intercept of a Scatchard plot generated in the
absence of competitor (not shown).) A 64-fold excess of fusion protein
over binding sites corresponds to approximately a 100-fold excess over
ribosomes at the constant input of ribosomes used in this experiment.
The fusion protein was serially diluted with elution buffer (5 mM glutathione, 50 mM Tris-HCl, pH 8.0), and
2.5-µl was added to each 20-µl reaction. The control sample
received 2.5 µl of elution buffer. Reactions were incubated on ice
for 10 min before addition of 10 eq of PKRM. The binding and flotation
steps were performed as in B. The fraction of ribosomes
bound was calculated as in B.
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Because the Sec61
c/GST fusion protein was able to
prevent the binding of ribosomes to stripped rough microsomes, we
suspected that the fusion protein might act by binding directly to
ribosomes at the site where the native Sec61
normally binds. We
therefore investigated the direct binding of labeled ribosomes to the
GST fusion proteins. The Sec61
c/GST fusion protein was
again expressed in bacteria and purified using glutathione-agarose
beads, but rather than eluting the fusion protein from the beads (as in
the competition experiment shown in Fig. 1), we tested the
Sec61
c/GST-coated glutathione-agarose beads as a solid
substrate for ribosome binding. By using different inputs of
SLR-labeled ribosomes, we obtained a Scatchard plot for binding to
Sec61
c/GST immobilized on glutathione-agarose, as shown
in Fig. 2 (open squares). The
affinity value (KD) obtained by this method was
1.2 × 10
8 M, a value
identical to that obtained by Borgese et al. (24) for the
binding of tritium-labeled nontranslating ribosomes to stripped rough
microsomes at low salt concentrations (25 mM KCl) and
within the range of values reported by Kalies et al. (25) for the binding of SLR-labeled nontranslating ribosomes to stripped rough microsomes at 25 and 150 mM KCl, respectively. We
emphasize that the very close correspondence between our affinity value for ribosome binding to Sec61
c/GST and measured values
for ribosome binding to stripped rough microsomes may be fortuitous,
given the inherent differences in the methods of measurement; however, we conclude that the affinity of ribosomes for the fusion protein is
roughly on the same order as their affinity for stripped rough microsomes. Glutathione-agarose beads coated with GST alone (Fig. 2,
closed squares) possessed a much lower ribosome binding
activity; this binding was unspecific, as indicated by the flat profile of the Scatchard plot. Based on the X intercept of the
Scatchard plot, we calculated that ~875 molecules of
Sec61
c/GST are required per ribosome bound. This high
figure may be accounted for by the additive effect of at least three
considerations as follows: 1) the ribosome may have multiple binding
sites for Sec61
, as evidenced by the estimate of 3-4 Sec61p
heterotrimers per translocon (17); 2) the large size of the ribosome
(Mr ~4.5 × 106) relative to
Sec61
c/GST (Mr ~3.2 × 104) means that a single bound ribosome may shield access
to many Sec61
c/GST molecules on the beads that are not
actively involved in binding; and 3) due to the porosity of the
glutathione-agarose, a large fraction of the Sec61
c/GST
may be bound in interior spaces that are not accessible to
ribosomes.

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Fig. 2.
The
Sec61 c/GST fusion protein
immobilized on glutathione-agarose beads binds directly to
ribosomes. The binding experiment was performed as described under
"Experimental Procedures" for the direct binding of ribosomes to
glutathione-agarose beads with the bound fusion protein. 20 µg of
Sec61 c/GST or GST alone, bound to glutathione-agarose
(~25 µl of beads), was used for each data point. A Scatchard plot
was obtained from measurements of the bound and unbound counts, and the
dissociation constant (KD) for binding of ribosomes
to Sec61 c/GST was obtained from the slope of the plot.
The total number of binding sites was estimated from the X
intercept, using the value given in the legend to Fig. 1 for the
molecular weight of the fusion protein.
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The cytoplasmic domain of Sec61
has a predicted isoelectric point of
12.0, with ~10% basic residues and a predicted net charge of +8 at
neutral pH. This raised the possibility that the ribosome binding
activity of Sec61
c/GST might result from a relatively unspecific charge-based interaction with ribosomal RNA. If this were
the case, yeast transfer RNA would be expected to compete efficiently
with labeled ribosomes for binding to Sec61
c/GST-coated glutathione-agarose beads. In Fig. 3,
yeast transfer RNA was added to the direct binding assay at a molar
excess of up to 2500-fold relative to inactive labeled ribosomes. Given
that the ratio of the masses of total ribosomal RNA to tRNA (per mol)
is ~125:1, an unspecific binding of ribosomal RNA should have been
reduced 40-50% by a 100-fold molar excess of tRNA, even if all the
ribosomal RNA was available for binding. In actuality a substantial
amount of the ribosomal RNA is likely to be buried within the particle (37), so an even greater reduction of ribosome binding might be
expected if the binding were simply charge-dependent.
Instead, the reduction in the binding of labeled ribosomes to
Sec61
c/GST does not approach 50% until a 2500-fold
molar excess of tRNA is added (Fig. 3, left panel).
Unlabeled ribosomes, however, compete efficiently (Fig. 3, right
panel).

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Fig. 3.
Yeast transfer RNA does not compete
efficiently for the direct binding of nontranslating ribosomes to
Sec61bc/GST. 3 µg of SLR-labeled ribosomes was incubated with
the indicated molar excess of yeast tRNA (suspended in RBB) or
unlabeled ribosomes before the addition of 20 µg of
Sec61 c/GST bound to glutathione-agarose. Volumes were
compensated with RBB. The binding reaction and subsequent analysis were
carried out as described in Fig. 2. RNA concentration was estimated by
A260. The Mr of RNA in
the ribosome was assumed to be 2.5 × 106, and the
average Mr of yeast tRNA was assumed to be
2 × 104. The fraction bound (y axis)
represents the number of bound counts relative to the sample with no
competitor, which was set equal to 1 in each panel.
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We also examined the salt dependence of ribosome binding to
Sec61
c/GST. Stripped rough microsomes have a large
ribosome binding capacity at low salt concentrations (25 mM
KCl); the fraction of ribosomes bound in vitro falls off
steeply as the salt concentration is increased to 0.5 M
(24). However, the affinity of binding is higher at a more
physiological salt concentration (150 mM KCl) than at 25 mM KCl (25). To examine the salt sensitivity of ribosome binding to the Sec61
c/GST fusion protein, we performed
the direct binding assay described above, at a range of salt
concentrations. As shown in Fig. 4, the
ribosome binding activity of Sec61
c/GST (open
squares) was stabilized by moderate salt concentrations and did
not fall off appreciably unless the KOAc concentration was raised from
250 to 500 mM, a concentration at which the dissociation of
nontranslating ribosomes from native rough microsomes is essentially complete (24). The base-line level of binding obtained with GST alone
(Fig. 4, closed squares) was insensitive to the salt concentration. The fact that the binding of ribosomes to
Sec61
c/GST, unlike the binding of ribosomes to stripped
rough microsomes, is actually favored at moderate versus low
salt concentrations, may be due to the absence, in the
Sec61
c/GST agarose beads, of low affinity interactions
that account for much of the binding to stripped rough microsomes at
low salt concentrations (25).

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Fig. 4.
Salt dependence of ribosome binding to
Sec61 c/GST. 3 µg of
SLR-labeled ribosomes was mixed with 20 µg of GST or
Sec61 c/GST bound to glutathione-agarose. Binding
reactions were carried out in RBB at the indicated KOAc
concentrations (25, 50, 100, 150, 250, 500, and 925 mM).
These KOAc concentrations were also used in the buffer added before the
separation step (see "Experimental Procedures"). Otherwise the
binding reaction and subsequent steps were carried out as in the legend
to Fig. 2.
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Ribosome binding to the ER membrane is believed to occur largely
through the large ribosomal subunit; in some reconstructions, however,
the subunits lie side-by-side on the membrane, and an interaction
between the small subunit and the membrane remains possible (38-40).
In the studies of Borgese et al. (24), isolated large
ribosomal subunits are capable of re-binding in vitro to stripped rough microsomes; small subunits also bind but to a lesser extent. We wished to test whether the Sec61
c/GST fusion
protein binds preferentially to the large ribosomal subunit, as is the case with native rough microsomes. A fixed amount of labeled ribosomal monomers (80 S) was added to a binding reaction containing
Sec61
c/GST-agarose beads
(Sec61
c/GST, Fig.
5, left panel) or stripped
rough microsomes (PKRM, Fig. 5, right panel). An
equimolar amount of unlabeled small (40 S) or large (60 S) subunits
or monomers (80 S) was added as a competitor, and the extent of
binding of the unlabeled component was inferred from the extent to
which the binding of labeled monomers was reduced. In both stripped
microsomes and Sec61
c/GST-agarose beads, unlabeled
monomers (80 S) could compete efficiently for binding. Small subunits
(40 S) had a more modest effect on binding. The effect of large
subunits (60 S) was greater in stripped rough microsomes than in
c/GST-agarose beads, but this difference may not be
significant in comparison to the effect of the other unlabeled
competitors. In the case of Sec61
c/GST, the competitive
activity of large subunits was consistently greater than that of small
subunits, implying that Sec
c/GST binds preferentially to
large subunits.

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Fig. 5.
Large ribosomal subunits compete more
efficiently than small subunits in an assay for ribosome binding to
Sec61 c/GST. SLR-labeled 80 S
ribosomes were mixed with an equimolar amount of unlabeled 80 S
monomers (80 S), small subunits (40 S), or large subunits (60 S).
The amount added was determined by the A260 of
each preparation, following the relative values given by Borgese
et al. (24). To this mixture was added a limiting amount of
Sec61 c/GST beads (left panel) or PKRM
(right panel). The binding reactions and subsequent
measurements were carried out as described under "Experimental
Procedures" for ribosome binding to Sec61 c/GST glutathione-agarose
beads (as in Figs. 2-4) and for ribosome binding to stripped membranes
(as in Fig. 1), respectively. Values in the graph are expressed as a
fraction of bound:unbound counts, relative to a sample where no
competitor was added (no comp.). 3-5 replicates were used
to obtain each data point shown in the graph.
|
|
 |
DISCUSSION |
By using two complementary methods, we have obtained results
suggesting physical contact between the Sec61
subunit and the ribosome. First, the Sec61
cytoplasmic domain
(Sec61
c) prevents ribosome attachment to ER membranes
when it is added to an in vitro ribosome binding assay in
the form of a GST fusion protein (Fig. 1). Second, the
Sec61
c/GST fusion protein binds directly to ribosomes
(Fig. 2), and the ribosome binding affinity of
Sec61
c/GST is comparable to that of stripped rough microsomes.
Our results are surprising in view of the work of Kalies et
al. (29) showing that Sec61
-depleted proteoliposomes bind to nontranslating ribosomes with an affinity comparable to that of proteoliposomes containing intact Sec61p. Based on this it appears possible that the ribosome binding activity of Sec61
, as detected in
our experiments, is merely redundant in vivo. However, the reported ribosome-binding affinities of the Sec61
-depleted and the
mock-depleted preparations (50 and 45 nM, respectively) in the latter study, although comparable to each other, are both considerably weaker than the affinities measured for ribosome binding
to native stripped microsomes in the work of the same authors (25, 41)
which agree with the values from our own experiments (not shown; see
also Ref. 24). As we did not attempt to reproduce the depletion and
reconstitution experiments in the present study, we cannot interpret
this discrepancy with any confidence. It nonetheless leaves room for
speculation that the effect of Sec61
depletion in the report by
Kalies et al. (29) is masked by a general reduction in the
affinity of both Sec61
-depleted and mock-depleted proteoliposomes
and that the contribution of Sec61
to in vitro ribosome
binding is more substantial than the latter results suggest. The
difference in ribosome binding affinity between ER-derived
reconstituted proteoliposomes and purified Sec61p-reconstituted
membranes also leaves open the possibility that, in the former case,
ribosome binding involves ER membrane proteins other than Sec61p. At
least two ER membrane proteins with independent ribosome binding
activity, p34 (42, 43) and p180 (44), have been identified previously,
although neither has been shown to associate with the translocon.
Binding of ribosomes to membrane proteins other than Sec61p in
reconstituted proteoliposomes might mask an effect of Sec61
depletion on the ribosome binding affinity of Sec61p itself.
The study of Kalies et al. (29) also shows that the
depletion of Sec61
impairs the in vitro transport of
preprolactin into Sec61-reconstituted membranes, although this
impairment can be overcome by allowing sufficient time for targeting of
the ribosome-nascent-chain complex before resumption of translation. In
the absence of a requirement for Sec61
in ribosome binding, the
authors (29) suggest that Sec61
might be involved in a
post-targeting step, possibly assisting in the insertion of the nascent
chain into the translocation pore. This agrees with observations by
Laird and High (45) that the nascent chain can be chemically
cross-linked to Sec61
at a point near its site of synthesis on the
ribosome, presumably before it enters the translocation pore. In our
study, the Sec61
c/GST fusion protein with high affinity
to nontranslating ribosomes suggests that the physiological role of
Sec61
is not limited to an interaction with the nascent chain.
However, our results are not incompatible with the idea that Sec61
normally acts only at a post-targeting stage. If the ribosome normally binds first to another membrane component and then to Sec61
, depletion of Sec61
from membranes should not affect their apparent ribosome binding activity; presentation of Sec61
out of its normal context (as in our study) would reveal its latent ribosome binding activity. This would reconcile our findings with those of Kalies et al. (29), where depletion of Sec61
from the Sec61p
complex did not affect its affinity for ribosomes. However, given that the cytoplasmic domain of Sec61
is large compared with the other cytoplasmic portions of the Sec61p complex, it seems unlikely that this
domain is normally blocked or buried within the complex. Consistent
with the idea that Sec61
is not essential for translocation, preliminary studies in our laboratory did not reveal an effect on the
translocation of preprolactin when the Sec61
c/GST fusion protein was added to a reticulocyte lysate translation system in excess
over ribosomes (not shown).
The question of whether Sec61
c/GST binds primarily
to ribosomal protein or to ribosomal RNA was not addressed in this
study. The identity of the ribosomal components involved in ER membrane binding has not been firmly established. Although previous studies identified groups of ribosomal proteins that are relatively
inaccessible to chemical modification in the membrane-bound
versus the free state (46, 47), the recent work of Prinz
et al. (41) indicates that the ribosome binds to the ER
membrane through the 28 S ribosomal RNA and that extensive proteolysis
of ribosomes does not affect their ability to bind ER membranes.
However, the type of ribosome binding measured in these experiments was
insensitive to the presence of Sec61
(29, 41), and it remains
possible that the binding of ribosomes to Sec61
reported here
involves a different cognate ribosomal component. The in
vitro binding of ribosomes to Sec61
c/GST is the
most reductive system available at present for the study of the
ER-ribosome junction, and further biochemical studies using this system
may avoid some difficulties inherent in studies using native rough microsomes.
 |
ACKNOWLEDGEMENTS |
The human Sec61
cDNA was given
by Dr. S. Prehn, Institute für Biochimie, Berlin, Germany.
pGEX-EF was given by Dr. D. Ron, Department of Cell Biology, New York
University School of Medicine. Anti-S3 antibody was given by Dr.
P. A. Marks and Dr. R. A. Rifkin, Cornell University Graduate
School of Medical Sciences, New York. Anti-L5 antibody and
Sec61p-reconstituted liposomes were given by Dr. R. Zimmermann,
Saarland University School of Medicine, Homburg, Germany. We thank Dr.
M. G. Rush, Department of Biochemistry, New York University School
of Medicine, for ideas and extensive discussion.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the American
Cancer Society and by the National Institutes of Health Medical Scientist Training Program.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.
¶
To whom correspondence should be addressed: Dept. of Cell
Biology, New York University School of Medicine, 550 1st Ave., New York, NY 10016. Tel.: 212-263-5317; Fax: 212-263-8139; E-mail: kreibg01@mcrcr.med.nyu.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M004867200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
SRP, signal recognition particle;
NAC, nascent-polypeptide-associated complex;
RM, rough microsomes;
PKRM, puromycin/high salt stripped rough microsomes;
PCR, polymerase chain
reaction;
GST, glutathione S-transferase;
SLR, sulfur
labeling reagent;
Ac, acetate anion.
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