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J Biol Chem, Vol. 275, Issue 3, 2037-2045, January 21, 2000
From the Department of Cell Biology, Duke University Medical
Center, Durham, North Carolina 27710
In this study, the contributions of
membrane-bound ribosomes to the regulation of endoplasmic reticulum
translocon composition and Sec61 Beginning with early morphological and biochemical studies
defining the rough endoplasmic reticulum as the site of protein entry
into the secretory pathway (1-3), an understanding of the mechanism of
ribosome binding to the endoplasmic reticulum
(ER)1 membrane has been
considered essential to the elucidation of the molecular mechanism of
protein translocation. A theme consistent through the past decades of
research into protein translocation has been the hypothesis that the
membrane-bound ribosome performs regulatory functions that govern the
structural and functional state of the translocon, the site of protein
translocation in the ER (4-9). In these models, the ribosome is
thought to interact with protein components of the ER membrane and
thereby initiate the assembly of a protein-conducting channel through
which translocation proceeds (5-8, 10, 11). In addition to a role in
the regulation of channel assembly, the ribosome is also thought to
regulate the structural state of the protein-conducting channel during the translocation event itself and in this manner assist the assembly of proteins of complex topologies, such as apolipoprotein B-100, the
prion protein, and polytopic membrane proteins (9, 12-14).
Diverse experimental approaches have provided evidence indicating a
role for the ribosome in the regulation of translocon assembly. For
example, following solubilization of rough microsomes (RM) with
digitonin, ribosomes co-fractionate with Sec61 Current experimental evidence supports the identification of Sec61 We report that solubilization of RM with digitonin yields
macromolecular complexes comprising ribosomes, a diverse population of
integral membrane proteins, and phospholipids. Such complexes were also
observed following solubilization of ribosome-free membranes, indicating that the ribosome is not required for complex stability in
detergent solution. In contrast to the results seen with digitonin, solubilization of RM with lysophosphatidylcholine or
1,2-diheptanoyl-sn-phosphatidylcholine (DHPC) yields
monodisperse solutions of RM proteins that did not reside in stable
association with ribosomes. In studies designed to assess the role of
Sec61 Reagents--
Digitonin was obtained from Wako Chemicals USA
(Richmond, VA). DHPC was from Avanti Polar Lipids (Alabaster, AL).
Puromycin and BigCHAP were obtained from Calbiochem (San Diego, CA).
5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-di- hydroxypropyl)-isophthalamide (Nycodenz), phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, and dinitrophenol were obtained from Sigma.
Chymotrypsin was from Worthington. Rabbit reticulocyte lysate was from
Promega (Madison, WI). [35S]Pro-Mix
([35S]methionine/cysteine) was obtained from Amersham
Pharmacia Biotech.
Preparation of Rough Microsomes, Ribosome-stripped Microsomes,
and Ribosomes--
Canine rough microsomes (RM), prepared as in Ref.
31, were stripped of ribosomes by treatment with EDTA and KOAc (EKRM), as described previously (30). Alternatively, microsomes were stripped
of ribosomes by treatment with puromycin and KOAc (PKRM) as described
previously (6). To prepare ribosomes, rabbit reticulocyte lysate (200 µl) was centrifuged for 30 min at 80,000 rpm in a TLA100 rotor
(Beckman Instruments, Palo Alto, CA) at 4 °C. The ribosomal pellet
was resuspended in 130 µl of ribosome buffer (25 mM
K-HEPES (pH 7.2), 50 mM KOAc, 5 mM
Mg(OAc)2, 1 mM DTT), loaded onto a 70-µl 0.5 M sucrose cushion of similar ionic composition, and
centrifuged as described above. Ribosomes were resuspended in ribosome
buffer, and concentrations determined by UV spectrometry (1 A260 unit = 21.4 pmol of 80 S ribosomes;
Ref. 32).
Analysis of Protein and Lipid Association with
Ribosomes--
Detergent solubilization of microsomal vesicles was
performed as follows: 10 equivalents (eq; defined in Ref. 31) of RM, or
an equal quantity of ribosome-stripped membranes, were suspended in
buffer containing 20 mM detergent (unless otherwise
specified), 25 mM K-HEPES (pH 7.2), 150 mM
KOAc, 5 mM Mg(OAc)2, 1 mM DTT, and
0.5 mM PMSF in a final volume of 200 µl. Samples were
incubated on ice 30 min, and ribosomes were pelleted by centrifugation
for 30 min at 80,000 rpm in a TLA100 rotor at 4 °C (247,000 × g at rav; k factor = 10). Supernatant fractions were precipitated by addition of
trichloroacetic acid to 10%, incubation on ice for 20 min, and
centrifugation for 15 min at 15,000 rpm in a refrigerated microcentrifuge. Pellets and precipitated supernatants were then processed for SDS-PAGE. SDS-PAGE gels were transferred to
nitrocellulose membranes using a semidry transfer apparatus (Bio-Rad)
and a transfer buffer consisting of 50 mM CAPS (pH 11.0),
20% methanol, and 0.075% SDS. Immunoblots were visualized using
SuperSignal detection reagents (Pierce), and images were quantified
using NIH Image software.
Velocity sedimentation studies of ribosome-associated membrane proteins
were performed by the following procedure; 30 eq of RM were suspended
in buffer containing 20 mM detergent, 25 mM K-HEPES (pH 7.2), 150 mM KOAc, 5 mM
Mg(OAc)2, 1 mM DTT, and 0.5 mM PMSF
in a final volume of 600 µl. Samples were incubated on ice for 30 min, and then loaded onto 10-ml 10-30% sucrose gradients containing
25 mM K-HEPES (pH 7.2), 150 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, and 0.1%
detergent. Gradients were centrifuged for 2 h at 40,000 rpm in an
SW40 rotor (Beckman) at 4 °C. Gradients were manually fractionated
by puncturing the tube bottoms, and fractions were precipitated by
addition of trichloroacetic acid to 10%. Samples were then processed
for immunoblot analysis as described above. To study interactions
between detergent-solubilized membrane proteins, the following
modifications were made; following solubilization of RM or
ribosome-stripped membranes, samples were centrifuged at 80,000 rpm for
30 min in a TLA100.2 rotor at 4 °C (228,000 × g at
rav; k factor = 18).
Supernatants were then loaded onto 10-ml 5-20% sucrose cushions
containing 25 mM K-HEPES (pH 7.2), 150 mM KOAc,
5 mM Mg(OAc)2, 1 mM DTT, and 0.1%
detergent. Gradients were centrifuged for 24 h at 40,000 rpm in an
SW40 rotor at 4 °C and processed as described above.
To study the association of bulk ER lipids with ribosomes, samples were
solubilized and fractionated through 10-30% sucrose gradients as
described above. The ribosome-enriched fractions were pooled and
diluted 4-fold with 25 mM K-HEPES (pH 7.2), 150 mM KOAc, 5 mM Mg(OAc)2, and 1 mM DTT. Ribosomes were pelleted by centrifugation for
4.5 h at 45,000 rpm in a Ti50.2 rotor (Beckman) at 4 °C.
Ribosomes were then resuspended in ribosome buffer, and ribosome-associated lipids were extracted and analyzed as described below.
Reconstitution of Vesicles from DHPC-solubilized RM
Extracts--
1000 eq of RM were pelleted and resuspended in 3 ml of
25 mM K-HEPES (pH 7.2), 150 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, 0.5 mM PMSF, 10% glycerol, and DHPC yielding a detergent:RM
ratio of approximately 400:1 (nanomoles of detergent:eq of RM). Samples were incubated on ice for 30 min and then centrifuged for 30 min at
80,000 rpm in the TLA100.2 rotor at 4 °C. Supernatant samples were
adjusted to 600 mM KOAc and mixed with SM-2 Biobeads
(Bio-Rad) that had been pre-equilibrated with 25 mM K-HEPES
(pH 7.2), 600 mM KOAc, and 10% glycerol. Samples were
incubated with shaking overnight at 4 °C. The fluid phase was then
removed from the beads, and the reconstituted vesicles were pelleted by
centrifugation for 10 min at 60,000 rpm in a TLA100.2 rotor at 4 °C.
Vesicles were resuspended in 0.25 M sucrose, 25 mM K-HEPES (pH 7.2), 50 mM KOAc, and 1 mM DTT and stored at Ribosome Binding Assays--
To analyze the binding of ribosomes
to reconstituted vesicles, 2 pmol of purified ribosomes were combined
with increasing quantities of vesicles in 50 µl of buffer consisting
of 25 mM K-HEPES (pH 7.2), 150 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, and 0.5 mM PMSF. Samples were incubated on ice for 30 min, and
Nycodenz was then added to a final concentration of 45%, to yield a
final volume of 1 ml while maintaining constant ionic conditions. The 45% Nycodenz-ribosome binding reaction was overlaid with 600 µl of
37.5% Nycodenz, 400 µl of 30% Nycodenz and 100 µl of 0%
Nycodenz, all in 25 mM K-HEPES (pH 7.2), 150 mM
KOAc, 5 mM Mg(OAc)2. Samples were centrifuged
for 3 h at 55,000 rpm in an SW55 rotor (Beckman) at 4 °C. The
upper 600 µl of the step gradients, which contained the floated
vesicles, was collected and precipitated by addition of trichloroacetic
acid to 10%. Samples were processed for SDS-PAGE and immunoblot
analysis as described above.
Proteolysis of Intact and Detergent-solubilized
Membranes--
Membranes were left untreated or solubilized with
digitonin or DHPC as described above, and chymotrypsin was added to the concentrations indicated in the figures. Digestions were performed for
30 min on ice, after which time trichloroacetic acid was added to 10%.
Samples were collected by centrifugation and processed for SDS-PAGE and
immunoblotting. When SDS was used to solubilize membranes, the above
procedures were carried out at 37 °C to avoid precipitation of the
SDS. In experiments involving ribosome binding to membranes prior to
proteolysis, 10 pmol of ribosomes were incubated with 2 eq of PKRM for
30 min at 4 °C in buffer containing 150 mM KOAc, 25 mM K-HEPES (pH 7.2), 5 mM Mg(OAc)2,
and 1 mM DTT.
Protein Translation Activity of Detergent-isolated
Ribosomes--
2 pmol of ribosomes, derived from either reticulocyte
lysate or RM solubilized with 20 mM DHPC, were added to 8 µl of a rabbit reticulocyte lysate post-ribosomal supernatant
supplemented with 500 ng preprolactin mRNA, 16 µCi of
[35S]Pro-Mix, 0.05 unit/ml RNasin, 1 mM DTT,
and 20 µM amino acid mix (minus methionine) in a total
volume of 20 µl. Following incubation for the indicated time periods
at 25 °C, 5 µl of the translation reaction was spotted onto filter
paper and protein synthesis levels were measured as described
previously (33).
Lipid Extraction and Analysis--
Samples were mixed with 4 volumes of a mixture of methanol/chloroform/HCl (100:50:1). After
extraction at room temperature for 20 min, phase separation was induced
by addition of 1.3 volumes each of chloroform and 2 M KCl.
Samples were centrifuged for 2 min at 13,000 rpm in a microcentrifuge.
The upper aqueous phase was removed, and the lower organic phase was
washed twice with a mixture of methanol/water/HCl (50:50:1). The
organic phase was reduced to dryness by vacuum centrifugation and
resuspended in chloroform/methanol (2:1). Bulk ER lipids were resolved
on silica thin layer chromatography (TLC) plates using a solvent system of chloroform/methanol/acetic acid/water (100:30:35:3) and were visualized by staining with iodine vapor.
Antibodies--
Anti-peptide rabbit polyclonal antisera against
Sec61 Composition of Detergent-soluble Ribosome-Translocon
Complexes--
To investigate the role of membrane-bound ribosomes in
the regulation of translocon composition, native RM were solubilized with either digitonin or DHPC, and ribosome-associated membrane components were resolved by sedimentation of the detergent-soluble fraction on sucrose gradients. The selection of detergents was based on
published studies identifying either ribosome-membrane protein
complexes (digitonin; Ref. 6) or high recovery of native protein
structure and activity (DHPC; Ref. 34). As depicted in Fig.
1 (A and C), when
RM were solubilized with digitonin, the ribosome fraction sediments as
a heterogeneous fraction containing Sec61 Translocon Structure Persists in the Absence of Bound
Ribosomes--
The data presented in Fig. 1 indicated that, when RM
were solubilized with digitonin, membrane-bound ribosomes sedimented in
association with a large, heterogeneous complex of integral membrane
proteins and phospholipids. When RM were solubilized with DHPC,
however, stable ribosome-membrane protein interactions were not
detected. To further investigate these differences, RM were stripped of
bound ribosomes in puromycin/high salt buffers (6) and experiments
similar to those depicted in Fig. 1 were performed (Fig.
2). In the absence of bound ribosomes,
the digitonin-soluble membrane proteins would be predicted to sediment
with a considerably smaller sedimentation coefficient than that
depicted in Fig. 1. Thus, to adequately resolve the detergent-soluble
protein fraction derived from ribosome-free membranes, the detergent
extracts were centrifuged on sucrose gradients for extended time
periods. As shown in Fig. 2A, when ribosome-free membranes
were solubilized with digitonin, Sec61
In current views, ribosome binding to the ER membrane is maintained
primarily through interactions with Sec61 Effects of DHPC on Oligomeric Protein Structure, Ribosome Binding,
and Ribosome Function--
DHPC is well documented to solubilize a
diverse array of integral membrane proteins while maintaining both
native structure and enzymatic activity (34, 35). Nonetheless, the
inability to detect stable translocon protein-ribosome interactions in
DHPC solution could result from DHPC-induced disruptions in the
structural integrity of ER oligomeric protein complexes, inactivation
of ribosome binding activity, and/or DHPC-induced structural
perturbations in the ribosome itself. To determine whether the
inability to detect ribosome-membrane protein interactions in DHPC
solution was a consequence of general disruptions in oligomeric protein integrity, the DHPC-soluble fraction from RM was fractionated by
velocity sedimentation and the oligomeric structure of the Sec61, TRAP,
and signal peptidase complexes determined. As shown in Fig.
3, the
The ribosome-Sec61
To determine if the lack of stable ribosome-membrane protein
interactions seen in DHPC solution were due to disruption of the
presumed electrostatic ribosome-membrane interaction by the choline
headgroup of DHPC, RM were treated with 0.5 M choline and
ribosome release was assayed. Under these conditions no ribosome release was observed (data not shown). In additional experiments, RM
were solubilized with digitonin and choline was subsequently added to
0.5 M. Again, ribosome-membrane component interactions remained intact (data not shown). These data indicate that the inability to detect stable ribosome-Sec61
To directly evaluate the effect of DHPC treatment on the ribosome
binding activity of microsomal membranes, the following experiments
were performed. In the first series of experiments, proteoliposomes
were reconstituted from DHPC-solubilized RM components and their
ribosome binding activity determined. Reconstituted vesicles were
incubated with ribosomes at physiological salt concentrations, and the
membrane-bound ribosomes separated by gradient flotation on Nycodenz
step gradients. Bound ribosomes were then quantified by immunoblotting
for the large ribosomal subunit proteins L3/L4. At maximal binding
stoichiometries, 2 pmol of ribosomes were bound to reconstituted
proteoliposomes containing the amount of Sec61
To assess whether DHPC elicited an alteration in ribosome structure, as
reflected in translation activity, the effects of DHPC on protein
synthesis were examined. In these experiments, a post-ribosomal
supernatant of reticulocyte lysate was supplemented with identical
quantities of ribosomes obtained either by centrifugation of
DHPC-solubilized RM, or resuspension of the reticulocyte ribosomes remaining from preparation of the lysate post-ribosomal supernatant. The capacity of the ribosome-supplemented post-ribosomal supernatant to
translate preprolactin was then assayed. As shown in Fig.
5B, the translation activity of the DHPC-treated ribosomes
was virtually identical to that seen with the reticulocyte ribosomes,
and thus DHPC does not alter ribosome structure in a manner that alters ribosome function. Furthermore, ribosomes recovered from
DHPC-solubilized RM are capable of binding to PKRM in a manner similar
to that depicted in Fig. 5A (data not shown).
Sec61
Similar experiments were then conducted to compare the Sec61
The protease sensitivities of Sec61
Previous studies demonstrating that the addition of inactive ribosomes
to PKRM elicits a protease-insensitive conformation of Sec61 In this study, the role of membrane-bound ribosomes in the
regulation of the macromolecular organization of the endoplasmic reticulum translocon and the conformation of Sec61 With regard to the role of the ribosome in regulating translocon
structure, it was observed that following solubilization of RM with
digitonin, ribosomes remained in association with large macromolecular
complexes containing Sec61 Previous studies have postulated that the interaction between the Sec61
complex and the ribosome occurs through stable electrostatic interactions (6). In support of this hypothesis, the release of
Sec61 To reconcile our experimental findings with previous studies, we
propose that the differences in the mechanism of membrane solubilization displayed by the two detergents determine whether ribosome association with the translocon can be captured. It is clear
that digitonin and DHPC solubilize biological membranes by different
mechanisms (39-43). Digitonin displays numerous structural similarities with the bile acid class of detergents. Principally, digitonin is a planar, heterocyclic nonionic detergent containing a
polyglycidic side chain. Previous studies have shown that detergents of
this structural class act by releasing membrane fragments enclosed at
their periphery with detergent molecules (44, 45). These studies also
noted that solubilization of membranes with such detergents preserves
the structural organization of the bilayer in the mixed micelle (44,
45). In contrast to digitonin, DHPC is a short-chain
phosphatidylcholine derivative, which, because of its pronounced
structural similarities to native membrane lipids and solubility in
aqueous media, efficiently disperses biological membranes (34, 46). In
so doing, DHPC is remarkable in its ability to preserve native membrane
protein structure and function in the solubilized state (34, 35). With
these differences in solubilization mechanism in mind, the data
presented herein can be interpreted to indicate that digitonin
treatment of native or stripped RM yields the release of large
macromolecular complexes constituting a defined mixed
detergent/protein/lipid domain. DHPC, because it is not subject to the
solubilization constraints imposed by the structural characteristics of
heterocyclic planar detergents such as digitonin, efficiently disperses
the protein and lipid components of the membrane bilayer (34). Thus,
whereas in digitonin the ribosome can maintain multiple contacts with
the membrane components that comprise the digitonin-soluble membrane
domain, in DHPC such membrane domains are not captured and thus stable ribosome-membrane protein interactions are not maintained. On the basis
of these data, we postulate that membrane-bound ribosomes maintain
their association with the ER membrane through multiple, weak
interactions, rather than via a high affinity bimolecular interaction.
In other words, the kinetic basis for ribosome binding to protein
components of the ER membrane is an avidity rather than an
affinity-based process. The hypothesis that ribosome binding to the ER
membrane is regulated through interactions involving multiple membrane
components is supported by reconstitution studies, where it was
observed that proteoliposomes containing the minimal translocation
machinery display approximately 10% of the ribosome binding observed
in native membranes, and by studies of the binding of biosynthetically
active ribosome nascent chain complexes, where ribosome binding to
sites other than Sec61 As an experimental approach, detergent-based co-fractionation studies
did not provide unequivocal data identifying a stable, stoichiometric
association between an ER translocon component(s) and membrane-bound
ribosomes, and so additional criteria were examined. As reported by
Kalies et al. (28), evidence for the identification of
Sec61 Because the protease-resistant conformation of Sec61 We thank Robyn Reed, Robert Seiser, Tianli
Zheng, and W. Hopper for helpful comments, support, and critical
reading of the manuscript.
*
This work was supported by National Institutes of Health
Grant DK47897 (to C. V. N.).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.
The abbreviations used are:
ER, endoplasmic
reticulum;
DHPC, diheptanoyl-sn-phosphatidylcholine;
RM, rough microsomes;
EKRM, EDTA- and high salt-washed rough microsomes;
PKRM, puromycin- and high salt-washed rough microsomes;
PAGE, polyacrylamide gel electrophoresis;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
CAPS, 3-(cyclohexylamino)propanesulfonic
acid.
Ribosome-independent Regulation of Translocon Composition and
Sec61
Conformation*
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DISCUSSION
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conformation were examined.
Following solubilization of rough microsomes (RM) with digitonin,
ribosomes co-sedimented in complexes containing the translocon proteins
Sec61, ribophorin I, and TRAP, and endoplasmic reticulum
phospholipids. Complexes of similar composition were identified in
digitonin extracts of ribosome-free membranes, indicating that
the ribosome does not define the composition of the
digitonin-soluble translocon. Whereas in digitonin solution a highly
electrostatic ribosome-translocon junction is observed, no stable
interactions between ribosomes and Sec61, ribophorin I, or TRAP
were observed following solubilization of RM with lipid-derived
detergents at physiological salt concentrations. Sec61 was found to
exist in at least two conformational states, as defined by mild
proteolysis. A protease-resistant form was observed in RM and
detergent-solubilized RM. Removal of peripheral proteins and ribosomes
markedly enhanced the sensitivity of Sec61 to proteolysis, yet the
readdition of inactive ribosomes to salt-washed membranes yielded only
modest reductions in protease sensitivity. Addition of sublytic
concentrations of detergents to salt-washed RM markedly decreased the
protease sensitivity of Sec61, indicating that a protease-resistant
conformation of Sec61 can be conferred in a ribosome-independent manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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, the core component
of the protein-conducting channel (6, 15). Furthermore, inactive
ribosomes elicit Sec61p oligomerization in proteoliposomes comprising
lipids and the purified Sec61p complex (7). In agreement with these
observations, a direct physical interaction between inactive yeast
ribosomes and yeast Sec61p was recently identified by cryo-electron
microscopy (8). It remains uncertain, however, whether the
Sec61p-ribosome interactions identified in these studies are identical
to the ribosome-membrane junction as characterized in vitro
(8, 16-19).
as the ribosome receptor, although substantial literature exists
regarding the identification of ribosome receptor proteins other than
Sec61 (20-29). Surprisingly, although significant disagreement regarding the identification and characterization of ribosome receptor
proteins persists, there is near unanimity in the choice of assay
system used for their discovery. In this assay, radiolabeled biosynthetically inactive ribosomes are incubated with
ribosome-stripped microsomal vesicles and the bound and free fractions
are separated by flotation centrifugation (20). This assay identifies
saturable and high affinity ribosome binding, although at physiological salt concentrations the observed ribosome binding stoichiometries are
approximately 10% of those found in native membranes (20, 28). For
this reason, it is uncertain whether the ribosome binding activity
observed in this assay is identical to that present during ribosome/nascent chain targeting and translocation. In fact, the hypothesis that ER membrane components other than Sec61 contribute to ribosome binding was presented in a recent study demonstrating the
binding of ribosomes bearing nascent secretory chains to sites on the
ER membrane other than Sec61 (30).
in the regulation of ribosome interactions with the ER
membrane, Sec61 was observed to exist in two conformational states.
In native RM and detergent extracts of native RM, Sec61 assumed a
protease-resistant conformation. Removal of bound ribosomes and
peripheral proteins yielded a dramatic increase in the sensitivity of
Sec61 to proteolysis; readdition of inactive ribosomes did not
efficiently restore the protease-resistant conformation. However, the
addition of sublytic concentrations of detergents to ribosome-depleted membranes markedly enhanced the resistance of Sec61 to proteolysis, indicating that Sec61 conformation can be regulated in a
ribosome-independent manner.
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DISCUSSION
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80 °C. The amounts of membrane proteins present in the vesicles, relative to RM, were determined by
immunoblotting of serial dilutions of reconstituted vesicles and
RM.
and 
, TRAP and , ribophorin I, and signal peptidase,
as well as chicken antisera (IgY) directed against ribosomal proteins
L3/L4, were used at 1:1500 dilution, as described previously (30). Following transfer to nitrocellulose, membranes were blocked in phosphate-buffered saline containing 5% nonfat milk and 0.1% Tween 20 for 1 h at room temperature. Primary incubations were performed for 1 h at room temperature in phosphate-buffered saline
containing 1% nonfat milk and 0.1% Tween 20, and secondary
incubations performed for 30 min in the identical buffer using the
appropriate secondary antibodies at 1:3000 dilution.
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, ribophorin I, TRAP,
and ER phospholipids. Quantitation of the ribosome-associated lipid
fraction by chemical phosphate analysis indicated that phospholipids
were present at approximately a 50-fold molar excess to ribosomes (data
not shown). When RM were solubilized with DHPC, none of the assayed
membrane components (Sec61, ribophorin I, TRAP, TRAM, or signal
peptidase complex) co-sedimented with ribosomes (Fig. 1B).
In addition, although phospholipids could be identified with the
DHPC-solubilized ribosomes (Fig. 1C), the quantities of
ribosome-associated phospholipids were approximately 2% of that seen
with digitonin-soluble ribosomes.
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Fig. 1.
Association of rough microsome components
with ribosomes in detergent solution. 30 eq of RM were solubilized
at 150 mM KOAc with 20 mM digitonin
(A) or DHPC (B) and the soluble fraction
subjected to velocity sedimentation in 10-30% sucrose gradients.
Gradient fractions were collected and processed for immunoblot analysis
with antibodies against the indicated proteins. The immunoblot for
L3/L4 indicates the position of ribosomes within the gradient. In
panel C, the ribosome peak from each gradient was
collected and the ribosomes recovered by centrifugation. Following
organic extraction of the pelleted ribosomes, the phospholipid
composition of the organic phase was determined by thin layer
chromatography and detection by iodine staining.
, ribophorin I, and TRAP
were again observed to migrate as a complex. Analysis of the total
protein composition of these fractions indicated that the
digitonin-soluble translocon complex was heterogeneous and consisted of
at least 20 silver stain-reactive polypeptides (Fig. 2B). In
contrast, when RM were solubilized with DHPC, Sec61, ribophorin I,
and TRAP exhibited overlapping, but non-identical sedimentation
profiles (Fig. 2C). From these experiments, it can be
concluded that the isolation of digitonin-soluble translocon complexes
does not require bound ribosomes. Furthermore, when RM were solubilized
with DHPC, the integral membrane protein components of the ER behaved
in a monodisperse manner and no stable membrane protein-ribosome
interactions were observed.
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Fig. 2.
Ribosome-independent macromolecular complexes
of ER membrane proteins exist in digitonin solution, but not in DHPC
solution. In panel A, a digitonin-soluble
PKRM extract was resolved on a 5-20% sucrose gradient. Fractions were
collected and subjected to immunoblot analysis for the indicated
proteins. In panel B, a digitonin-soluble PKRM
extract was resolved as in A, gradient fractions were
collected, the proteins were concentrated by trichloroacetic acid
precipitation, and following resolution on a 12.5% SDS-PAGE gel, the
protein components were identified by silver stain detection. A digital
image of the silver-stained gel is shown. In panel
C, a DHPC-soluble RM extract was processed as in
panel A, except smaller fraction sizes were
collected to obtain higher resolution.
(7, 28). The data depicted
in Figs. 1 and 2 support this view, although the molecular
heterogeneity of the digitonin-soluble translocon complexes, as seen in
both the presence and absence of bound ribosomes, prevents unequivocal
identification of Sec61 as the ribosome receptor. Alternatively, the
results obtained with DHPC can be interpreted to indicate that neither
Sec61, ribophorin I, nor TRAP alone constitute the primary site
of ribosome-membrane interaction. To reconcile these conflicting
interpretations, additional experiments were performed to critically
assess the significance of the DHPC data and to further evaluate
ribosome-Sec61 interactions.
and subunits of Sec61
undergo partial dissociation in DHPC solution, whereas other membrane
protein complexes, such as the TRAP and signal peptidase complexes,
remain wholly intact. From these data, it can be concluded that DHPC
does not elicit a general disruption of oligomeric membrane protein
complexes. Furthermore, because it has been demonstrated that the and subunits of Sec61 readily dissociate in detergent solution and that the subunit of Sec61 is not required for ribosome binding (36), the partial dissociation of the and subunits of Sec61 observed in DHPC solution does not provide a convincing explanation for
the lack of stable membrane protein-ribosome interactions in
DHPC-solubilized membranes.
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Fig. 3.
Effects of DHPC on membrane protein
quaternary structure. RM were solubilized at physiological salt
concentrations in 20 mM DHPC and the soluble fraction
resolved by velocity sedimentation in sucrose gradients as described in
the legend to Fig. 1. The oligomeric structures of the Sec61 complex,
the TRAP complex, and the signal peptidase complex were determined by
immunoblot analysis of the SDS-PAGE resolved gradient fractions.
interaction, as assayed in digitonin solution, is
highly electrostatic and is stable at salt concentrations approaching 1 M (6). Consistent with this observation, when native RM
were solubilized in digitonin at 0.5 M salt and
centrifuged, Sec61 was recovered in the ribosome-rich pellet
fraction, whereas when ribosome-stripped membranes were solubilized
with digitonin, Sec61 was recovered in the supernatant fraction
(Fig. 4A, lanes 5 and 6). In contrast, solubilization of native
RM with either DHPC or lysophosphatidylcholine at physiological salt
concentrations yielded efficient recovery of Sec61 in the
supernatant fraction (Fig. 4B, lanes
3-6) and, as illustrated in Fig. 4C, the
DHPC-soluble Sec61 was not associated with ribosomes. This
phenomenon is further illustrated in Fig. 4D, where the
ability of the two detergents to release Sec61 from native RM was
assayed as a function of detergent concentration. As is clear from the
data in Fig. 4D, whereas solubilization of Sec61 with
DHPC exhibits a clear maxima at a DHPC concentration of 16-24
mM (detergent:RM of 400-600 nmol detergent:eq RM, with 1 eq RM containing ~4 nmol of phospholipid), solubilization of Sec61
with increasing concentrations of digitonin does not alter the
distribution of Sec61 into large (>100 S) complexes that are
recovered in a ribosome-rich pellet upon centrifugation. Thus, if
ribosome association with the ER membrane is conferred via
electrostatic interactions with Sec61, such interactions are
disrupted in the presence of DHPC. Alternatively, ribosome binding to
the ER membrane may require multiple weak interactions with ER membrane
components and, under conditions in which the ER membrane is
efficiently solubilized (addition of DHPC or lysophosphatidylcholine), weak bi-molecular interactions between ribosomes and individual membrane components are not captured.
View larger version (23K):
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Fig. 4.
Detergent-mediated solubilization of
Sec61 in native microsomes and
ribosome-stripped microsomes. In panel A, 10 eq of RM or EKRM were solubilized with 20 mM digitonin at
the indicated KOAc concentrations. Ribosomes were pelleted by
centrifugation at 80,000 rpm for 30 min in the TLA100 rotor. The
pellets and supernatants were then subjected to immunoblot analysis
with an anti-Sec61 antibody. In panel B, 10 eq
of RM were solubilized with 20 mM lysophosphatidylcholine
or DHPC, and were processed as in panel A. In
panel C, 10 eq of RM were solubilized with 20 mM DHPC, and were processed as in panel
A, with the exception that antibodies recognizing ribosomal
proteins L3/L4 and S9 were used in immunoblot analysis. A similar
distribution of ribosomal components was observed when RM were
solubilized with digitonin or lysophosphatidylcholine. In
panel D, the efficiency of Sec61
solubilization from RM was assayed as a function of detergent
concentration at physiological salt concentrations. Note that RM
contain ~4 nmol of phospholipid/eq.
interactions following solubilization with DHPC was not solely due to the choline headgroup, and, because lysophosphatidylcholine treatment of RM also yielded soluble, ribosome-free Sec61 at moderate salt concentrations, not a
unique consequence of the relatively short (7 carbon) acyl chains
present on DHPC.
found in 3-4 eq of
RM (Fig. 5A). Native RM
contain approximately 1 pmol of bound ribosomes/eq (data not shown),
and so proteoliposomes prepared from DHPC extracts of RM display
approximately 50% of the ribosome binding activity observed in native
membranes. A 50% relative binding stoichiometry is consistent with a
stochastic reconstitution of Sec61 in the proper and inverse
topologies. Thus, solubilization of RM in DHPC and subsequent
reconstitution of the detergent extract into proteoliposomes does not
substantially alter the binding of inactive ribosomes.
View larger version (24K):
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Fig. 5.
Effects of DHPC on ribosome binding and
protein translation activity. In panel A,
vesicles containing the indicated amounts (eq) of Sec61 were
incubated with 2 pmol of ribosomes and were then subjected to flotation
through Nycodenz cushions as described under "Experimental
Procedures." Vesicles were harvested and subjected to immunoblot
analysis with antibodies recognizing the large ribosomal subunit
proteins L3/L4 (lanes 3-11). As controls,
unfloated vesicles were immunoblotted with the anti-L3/L4 antibody to
confirm the absence of ribosomal contamination (lane
1), and 2 pmol of ribosomes were immunoblotted with the
anti-L3/L4 antibody to indicate the total amount of ribosomes present
in the assay (lane 2). In panel
B, reticulocyte ribosomes (
), ribosomes obtained after
solubilization of RM with 20 mM DHPC at 150 mM
KOAc (
), or a control sample lacking ribosomes (×) were used in
cell-free translation assays, as described under "Experimental
Procedures." Following translation of full-length preprolactin
mRNA for the indicated time periods, samples were processed to
determine the amount of radioactive methionine incorporated into
trichloroacetic acid-insoluble protein.
Exists in Multiple Structural States Differing in Protease
Accessibility--
Although the results depicted in Fig. 5 support the
conclusion that solubilization of RM with DHPC preserves inactive
ribosome binding activity upon reconstitution, it was necessary to
address the hypothesis that Sec61, in DHPC solution but not in
digitonin solution, assumed a conformation unsuitable for ribosome
binding. To test this hypothesis, experiments were performed to examine the conformation of Sec61 in the detergent-solubilized state, using
the proteolysis pattern of Sec61 as an index of conformation. To
generate a conformation-sensitive proteolysis profile for Sec61, RM
were treated with increasing concentrations of SDS at 37 °C and
subjected to chymotrypsin digestion. As shown in Fig.
6A, in the absence of SDS,
Sec61 is relatively insensitive to proteolysis by chymotrypsin, with
the majority of the protein being recovered as the full-length form and
a minor fraction being recovered as a faster migrating (~28 kDa)
form, indicated by the double asterisk (Fig.
6A, lane 1). At low concentrations of
SDS, the conformation of Sec61 was significantly altered. Under
these conditions, the N-terminal epitope recognized by the Sec61
antisera was degraded and thus no limit digestion products were
detected (Fig. 6A, lanes 2-5).
However, at higher concentrations of SDS, where the chymotrypsin itself
becomes inactivated, the faster migrating limit digestion product was
readily detected (Fig. 6A, lanes
6-8). At the highest SDS concentration tested, in which the
protease was completely inactivated, Sec61 was recovered
largely in the intact form (Fig. 6A, lane
9).
View larger version (38K):
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Fig. 6.
Proteolysis of Sec61
in intact and detergent-solubilized RM and PKRM. In
panel A, 10 eq of RM were solubilized with the
indicated concentrations of SDS for 30 min at 37 °C. Chymotrypsin
was then added to a final concentration of 25 µg/ml, and digestion
occurred for 30 min at 37 °C. Digestion was stopped by addition of
trichloroacetic acid to 10%. Samples were then processed for
immunoblot analysis with an anti-Sec61 antibody. In panel
B, for lanes 1 and 2 and
lanes 7 and 8, 10 eq of RM or PKRM
were either untreated or subjected to proteolysis at 4 °C for 30 min
with chymotrypsin at a final concentration of 25 µg/ml. For
lanes 3-6 and 9-12, RM or PKRM were
first solubilized with 20 mM digitonin or DHPC at 150 mM KOAc, and were then subjected to proteolysis as
described above. Relative molecular weights are indicated to the
left of the panels. Sec61 limit digestion products are
indicated by single and double
asterisks.
digestion pattern obtained in native and ribosome-stripped RM, prior
and subsequent to solubilization with digitonin or DHPC (Fig.
6B). As demonstrated previously, in native RM, Sec61 was nearly completely insensitive to digestion by exogenous proteases (Fig.
6, A, lane 1; B,
lanes 1 and 2). In light of the
observation that ribosomes remain bound to Sec61 following
solubilization of RM with digitonin, the digitonin-soluble Sec61
fraction was predicted to be insensitive to protease digestion, and
this was observed (Fig. 6B, lanes 3 and 4). On the basis of the data presented in Fig. 4, we
postulated that in a DHPC extract of RM, Sec61, which is not in
association with ribosomes, would be protease-sensitive. This
prediction was not met. Rather, the proteolysis pattern of Sec61 in
DHPC solution was identical to that observed in digitonin solution
(Fig. 6B, lanes 5 and 6 versus lanes 3 and 4).
Thus, when solubilized from RM, Sec61 exists in a protease-resistant conformation regardless of whether it is (digitonin solution), or is
not (DHPC solution), associated with ribosomes. These data indicate
that the protease resistance of Sec61 in native membranes cannot be
attributed solely to bound ribosomes and furthermore in native RM
Sec61 exists in at least two conformations, which differ in their
relative sensitivity to chymotrypsin digestion.
in detergent extracts of
ribosome-stripped membranes (PKRM) were identical (Fig. 6B, lanes 9-12), yet distinct from those observed in
intact PKRM (Fig. 6B, lanes 7 and
8). In PKRM, virtually the entire population of Sec61 had
been converted to the chymotrypsin sensitive form, and was recovered as
the 28-kDa form (Fig. 6B, lanes 7 and
8). In contrast, when PKRM were solubilized with either
digitonin or DHPC, a third limit digestion product was identified. In
this conformational state, Sec61 was cleaved to yield a form of
intermediate mobility to the full-length and 28-kDa forms, and is
indicated by the single asterisk (Fig.
6B). Because the antisera used in these experiments was
raised against an N-terminal epitope, the mobility of this form on
SDS-PAGE is consistent with proteolysis occurring in a domain of
Sec61 C-terminal to that generating the 28-kDa limit digestion
product. As will be demonstrated in Fig.
7, this digestion product is likely an
intermediate in the formation of the 28-kDa form. In summary, the data
presented in Fig. 6 indicate that: 1) the conformation of Sec61 in
digitonin solution is similar to that observed in DHPC solution; 2)
removal of bound ribosomes and peripheral proteins elicits a
conformational change in Sec61; 3) the inability of proteases to
cleave Sec61 in native membranes cannot be attributed solely to
bound ribosomes, and 4) Sec61 can exist in at least two
conformations, both of which are present in native RM.
View larger version (56K):
[in a new window]
Fig. 7.
Modulation of Sec61
conformation. Panel A, RM, PKRM, or
PKRM supplemented with a 5-fold molar excess of inactive ribosomes in
physiological salt buffer were treated with increasing concentrations
of chymotrypsin (CT) for 30 min on ice. Proteolysis
reactions were terminated by addition of trichloroacetic acid. Samples
were resolved by SDS-PAGE and the Sec61 proteolysis pattern
determined by immunoblot analysis with an antibody directed against the
N terminus of Sec61. Panel B, PKRM, either
untreated or treated with 0.75 mM DHPC for 30 min on ice,
were proteolyzed with increasing concentrations of chymotrypsin and
processed for immunoblot analysis as described above. Panel
C, PKRM were treated with the indicated concentrations of
DHPC for 30 min on ice and centrifuged for 20 min at 80,000 rpm in a
TLA100 rotor at 4 °C. Pellet samples were resuspended in SDS-PAGE
sample buffer and processed by immunoblot analysis for BiP
content.
support
the conclusion that ribosome binding to Sec61 is the primary
determinant governing protease accessibility (28). As the data
presented in Fig. 6 indicate that the conformational status of Sec61
can be altered in a ribosome-independent manner, the effects of
ribosome addition on the conformation of Sec61 were investigated. In
these experiments, protease titrations were performed on RM, PKRM, and
PKRM to which excess inactive ribosomes were added (Fig.
7A). Comparison of the proteolysis patterns indicates that,
as demonstrated previously, Sec61 in RM exists in two conformational states, with the predominant form being resistant to chymotrypsin cleavage (Fig. 7A, lanes 1-5).
Addition of increasing concentrations of chymotrypsin to PKRM results
in the appearance of two forms: one of intermediate mobility, which is
identical to that seen upon digestion of detergent-solubilized PKRM
(Fig. 6B, lanes 10 and 12,
single asterisk), and one of higher mobility
(Fig. 6, A and B, double
asterisk), which is of identical mobility to that seen in
low abundance in native RM (Fig. 7A, lanes
6-10). When chymotrypsin titrations were performed on PKRM
supplemented with excess ribosomes, the overall pattern of digestion
was similar to that seen in the absence of ribosomes (Fig.
7A, lanes 7-10 versus
lanes 12-15). Comparison of the proteolysis
patterns at intermediate concentrations of chymotrypsin did indicate,
however, a modest influence of the inactive ribosomes on the
conformation of Sec61, primarily with regard to the intermediate
digestion product seen most readily in detergent solubilized PKRM (Fig. 7A, compare lanes 8 and 9 with lanes 13 and 14). Clearly,
however, the conformation of Sec61 in PKRM supplemented with a
5-fold molar excess of free ribosomes is markedly different from that present in RM. Additional experiments provided evidence for a ribosome-independent regulation of Sec61 conformation. As shown in
Fig. 7B, following treatment of PKRM with a sublytic
concentration of DHPC, Sec61 was markedly resistant to proteolytic
digestion (Fig. 7B, lanes 1-7
versus lanes 8-14). At the
concentration of DHPC used in these experiments (0.75 mM),
integral membrane proteins, such as Sec61, TRAM, and the signal
peptidase complex were not solubilized (data not shown), and lumenal
proteins such as BiP were not released from the membrane vesicles (Fig.
7C). Because the protease accessibility characteristics of
Sec61 as seen in intact RM were displayed upon addition of sublytic
concentrations of detergent to ribosome stripped membranes, it appears
that low concentrations of detergent either alter membrane structure in a manner that effects a change in Sec61 conformation and/or alter protein-protein interactions that contribute to the regulation of
Sec61 conformation. The observed DHPC-dependent changes
in Sec61 protease accessibility could also be obtained by addition of a structurally unrelated detergent (BigCHAP) or a compound that
preferentially distributes in the outer membrane leaflet (dinitrophenol), indicating that this phenomenon is not unique to DHPC
(data not shown). These data clearly demonstrate that the conformation
of Sec61, as displayed by limited proteolysis, can be modulated in a
ribosome-independent manner.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were
investigated. Six conclusions can be drawn from these studies. 1) In
digitonin solution, the translocon is a heterogeneous complex
consisting of multiple integral membrane proteins, phospholipids and
ribosomes. 2) The molecular composition of such complexes is
ribosome-independent. 3) When solubilized under conditions in which ER
membrane proteins are recovered in monodisperse solution, membrane
protein-ribosome interactions are not maintained. 4) Sec61 can be
found in at least two conformational states. 5) The proteolytic
sensitivity of Sec61 is enhanced upon removal of peripheral proteins
and bound ribosomes. 6) Conversion of Sec61 to a protease-resistant conformation, as is predominantly found in native RM, can be elicited by sublytic concentrations of detergent and with very modest efficiency by inactive ribosomes.
, ribophorin I, TRAP, and approximately
a 50-fold molar excess of phospholipid to ribosomes. Related studies by
Chevet et al. (37) indicate that similar detergent-derived
complexes also contain calnexin and the ER-to-Golgi recycling protein
2p24. The isolation of such macromolecular complexes is in agreement
with previous studies (48), and was not dependent on bound ribosomes,
as complexes of similar composition were obtained following
solubilization of ribosome-free microsomes (PKRM) with digitonin.
Because the digitonin-soluble translocon complexes are heterogeneous
and apparently not dependent upon the ribosome for their higher order
structure, it is difficult to determine whether the ribosome binding
activity displayed by such complexes reflects the activity of any
single component of the complex, as would be expected from
published data (6, 8, 15, 28, 38).
from digitonin-solubilized ribosome pellets required salt
concentrations in excess of 1 M (6). However, when RM were
solubilized with DHPC or lysophosphatidylcholine, no stable interactions between ribosomes and Sec61 were observed. Rather, following solubilization in DHPC at physiological salt concentrations, resident integral membrane proteins behaved in a monodisperse manner
and were readily resolved from membrane-derived ribosomes. This
observation was unexpected and thus necessitated a series of control
experiments to evaluate the effects of DHPC treatment on the structure
and activity of the two assumed binding partners, ribosomes and
Sec61. In these experiments, it was demonstrated that DHPC did not
elicit a general disruption of membrane protein quaternary structure,
that ribosome binding activity could be efficiently recovered in
proteoliposomes reconstituted from DHPC extracts of RM, that the
protease accessibility of Sec61 in either digitonin or DHPC extracts
was similar, and that DHPC treatment did not alter the protein
translation activity or membrane binding activity of ribosomes.
Solubilization of RM with DHPC does not, therefore, appear to alter the
structural or functional characteristics of the ER components that are
presumed responsible for ribosome binding. It should be noted, however,
that although extensive control experiments indicated that
solubilization of RM with DHPC did not irreversibly affect ribosome
binding, we cannot eliminate the possibility that DHPC elicits a
heretofore uncharacterized conformational change in Sec61 that
disrupts Sec61-ribosome interactions. Equally as important, the
substantial molecular heterogeneity of the digitonin-soluble translocon
complexes and the observation that translocon composition in
digitonin is ribosome-independent makes the identification of the
ribosome receptor(s) in digitonin extracts of RM difficult.
was reported (28, 30). Proteoliposomes
containing the SRP receptor and Sec61 function in translocation, and
so Sec61 likely figures prominently in the regulation of ribosome
binding (15).
as the ribosome receptor was obtained in studies demonstrating
that Sec61 is protected from proteolysis by membrane-bound
ribosomes, and that the addition of inactive ribosomes to
ribosome-stripped microsomes restores the protease resistance of
Sec61. To further examine this conclusion, the protease sensitivity
of Sec61 was compared in native membranes, native membranes
solubilized with digitonin (where a stable ribosome-membrane junction
is maintained), and native membranes solubilized with DHPC (where the
ribosome-membrane junction is lost). Contrary to prediction, the
protease sensitivity of Sec61 did not increase following
solubilization with DHPC. If native membranes were stripped of bound
ribosomes with high salt and puromycin prior to solubilization and
proteolysis, however, Sec61 was readily accessible to chymotrypsin digestion. These data indicate that the conditions used to extract membrane-bound ribosomes from ER microsomes alter Sec61 protease accessibility, but as the data obtained with DHPC extracts depicts, the
protease-resistant conformation seen in native membranes need not be
conferred by the bound ribosome. Furthermore, when ribosome-stripped membranes are supplemented with free ribosomes, only a very modest restoration of protease resistance is observed. From these data, it
appears that Sec61 can assume at least two conformations and that
the removal of peripheral proteins and bound ribosomes alters Sec61
conformation in a manner that is only inefficiently reversed by the
addition of excess inactive ribosomes. It follows that the binding of
inactive ribosomes to ribosome-stripped membranes is mechanistically
different from that occurring during the physiological targeting event
and so inactive ribosomes, should they bind to Sec61, do not protect
it from proteolysis.
could be
efficiently elicited by addition of sublytic concentrations of
detergent (Fig. 7) or the amphiphile dinitrophenol (data not shown), we
speculate that the differences in Sec61 protease accessibility seen
following the extraction of peripheral proteins represent, at least in
part, a conformational change in the protein. The observation that
Sec61 conformation is sensitive to the addition of sublytic
concentrations of amphiphiles suggests that the physical state of the
membrane can influence Sec61 conformation. When viewed with respect
to previous studies demonstrating that membrane-bound ribosomes
increase the structural order parameters of microsomal membranes (47),
these data identify a mechanism whereby membrane-bound ribosomes, by
influencing the physical properties of the membrane, could elicit
changes in Sec61 conformation in the absence of direct, stable interactions.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Cell Biology,
Box 3709, Duke University Medical Center, Durham, NC 27710. Tel.:
919-684-8948; Fax: 919-684-5481; E-mail:
c.nicchitta@cellbio.duke.edu.
ABBREVIATIONS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Sabatini, D. D.,
and Kreibich, G.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
9931-9935
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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