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J. Biol. Chem., Vol. 275, Issue 43, 33820-33827, October 27, 2000
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From the Department of Cell Biology, Duke University Medical
Center, Durham, North Carolina 27710
Received for publication, May 23, 2000
Contemporary models for protein translocation in
the mammalian endoplasmic reticulum (ER) identify the termination of
protein synthesis as the signal for ribosome release from the ER
membrane. We have utilized morphometric and biochemical methods to
assess directly the fate of membrane-bound ribosomes following the
termination of protein synthesis. In these studies, tissue culture
cells were treated with cycloheximide to inhibit elongation, with
pactamycin to inhibit initiation, or with puromycin to induce premature
chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained
membrane-associated. Analysis of the subunit structure of the
membrane-bound ribosomal particles remaining after termination was
conducted by negative stain electron microscopy and sucrose gradient
sedimentation. By both methods of analysis, the termination of protein
synthesis on membrane-bound ribosomes was accompanied by the
release of small ribosomal subunits from the ER membrane; the majority
of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER
membrane is regulated independently of protein translocation.
The endoplasmic reticulum
(ER)1 membrane, the site of
nascent secretory and integral membrane protein translocation, contains an abundance of membrane-bound ribosomes. As is now well established, the association of biosynthetically active ribosomes with the ER
membrane occurs through the activity of the signal recognition particle/signal recognition particle receptor targeting machinery (1-3). By this process, ribosome-nascent chain complexes
bearing secretory or membrane protein precursors are recognized early in synthesis and are trafficked from the cytosol to the ER membrane (4). Following the binding of the ribosome-nascent chain complexes to
the resident translocon complex of the ER membrane, protein translation
proceeds and nascent chains are translocated across or integrated into
the ER membrane. Subsequently, the termination of protein synthesis is
thought to elicit the release of the ribosomal subunits from the ER
membrane to the free, cytoplasmic pool (5, 6). In the cytoplasmic pool,
the ribosomal subunits are free to participate in the protein synthesis
initiation sequence. Should they engage in the synthesis of a secretory
or membrane protein precursor, targeting to the ER membrane again
occurs, thus describing a cycle of ribosome binding and release.
In the pioneering studies on ribosome-membrane interactions in the ER,
it was observed that ribosomes bind asymmetrically, with the binding
interaction being mediated entirely through the large subunits (7-10).
Thus, if isolated ER microsomes were extracted with increasing
concentrations of EDTA, the small ribosomal subunit was preferentially
released from the membrane, whereas the large ribosomal subunit
remained membrane-bound (7, 8). Confirmation of the nature of ribosome
binding to the ER was later obtained by direct ultrastructural analysis
of intact cells, where it was observed that the large ribosomal subunit
contains the site of membrane attachment (10). On the basis of these
observations, it may be predicted that the termination of protein
synthesis by membrane-bound ribosomes would be accompanied by the
regulated dissociation of large ribosomal subunits from the ER membrane.
Do ribosomes cycle between a membrane-bound and free state? This
question was first addressed in biosynthetic labeling and exchange
studies and yielded differing and somewhat contradictory conclusions
(9, 11). In the study of Baglioni et al. (9), it was
proposed that large ribosomal subunits bind to the ER membrane prior to
their assembly into polysomes. In contrast, Mechler and Vassalli (11),
using a similar isotope incorporation protocol, concluded that small
and large ribosomal subunits enter the membrane-bound polysome fraction
from the cytosolic pool at similar rates. Although these studies are in
disagreement regarding the entry path for large subunits into
membrane-bound polysomes, two points are evident. 1) The biosynthetic
labeling and exchange kinetics of membrane-bound large subunits differ
from those of membrane-bound small subunits. 2) Membrane-bound and
cytosolic ribosome pools constitute a common population. However,
because ribosome synthesis and assembly are relatively slow and complex
processes, the resolution offered by ribosome biosynthetic labeling
studies is not sufficient to analyze directly the temporal coupling
between protein synthesis on membrane-bound ribosomes and ribosome
exchange between free and bound ribosome pools.
The purpose of this study was to investigate experimentally the
compartmental fate of membrane-bound ribosomes following the termination of protein synthesis. Do ribosomes dissociate from the ER
membrane coincident with the termination of protein synthesis? By using
biochemical and morphometric methods in tissue culture cells, we report
that in vivo termination yields the preferential release of
ribosomal small subunits from the membrane, whereas the large ribosomal
subunits remain predominantly in the bound state.
Reagents--
35S-Labeled Pro-Mix
([35S]methionine and -cysteine) and
5,6-[3H]uridine were obtained from Amersham Pharmacia
Biotech. Pactamycin was obtained from the Upjohn Co. Cell culture media
and reagents were from Life Technologies, Inc. Digitonin was from Wako
Biochemicals (Richmond, VA). Antibodies to human albumin were from
Roche Molecular Biochemicals. Reagents for electron microscopy were
obtained from Electron Microscopy Sciences (Fort Washington, PA). All
other reagents were from Sigma.
Cell Culture--
Human hepatocarcinoma cells (HepG2) and
Buffalo Rat liver cells (BRL 3A) were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum at
37 °C and 5% CO2. Cells were subcultured at 3-day intervals.
Analysis of Protein Synthesis Inhibitor Activity--
Cells were
plated at 1 × 106 cells/well on 6-well plates and
used at 70% confluency. For pulse labeling studies, cells were first
starved of methionine by incubation in serum- and methionine-free DMEM
for 20 min at 37 °C. Pulse labeling was subsequently initiated by
replacement of the media with serum-free and methionine-free DMEM
supplemented with 100 µCi/ml 35S-labeled Pro-Mix. Where
indicated, protein synthesis inhibitors were added directly to the
pulse medium to final concentrations of 200 µM
cycloheximide, 200 µM puromycin, or 0.2 µM
pactamycin. After the indicated chase periods, cells were lysed in
phosphate-buffered saline containing 20 mM CHAPS and 10 mM EDTA and centrifuged (15 min, 15,000 × g) to remove insoluble cellular debris. Albumin was
immunoprecipitated from the cell lysate supernatant and media fractions
with rabbit anti-human albumin antibody and protein A-conjugated
Sepharose beads. Immunoprecipitates were processed for SDS-PAGE and
separated on 12.5% gels. The incorporation of radiolabel into albumin,
expressed in PSL units, was determined by phosphorimager
analysis with a MacBAS-1000 PhosphorImager system (Fuji Medical
Systems, Stamford, CT).
Electron Microscopy--
HepG2 and BRL cells were plated on
plastic coverslips in multiwell plates and grown to 70% confluency.
Alterations in protein synthesis activity were elicited by addition of
cycloheximide, puromycin, or pactamycin to the medium and incubation
for 10 min at 37 °C. The medium was then removed and replaced with
ice-cold 0.1 M sodium cacodylate buffer containing 3%
glutaraldehyde and 0.05% tannic acid. The samples were then fixed in
1% osmium tetroxide, stained in 2% uranyl acetate, dehydrated in a
graded alcohol series, and embedded in Araldite 506 epoxy resin.
60-80-nm ultrathin sections were cut with a diamond knife on an
ultramicrotome, collected on coated copper grids, and stained with
permanganate and lead citrate (12). Grids were viewed at 60 kV in a
transmission electron microscope at magnifications of 15,000-120,000
(JEOL, Peabody, MA).
For negative staining, BRL cells were grown in normal media and treated
with cycloheximide, pactamycin, or puromycin as described above. After
harvesting with trypsin/EDTA, the cells were washed and resuspended to
5 × 106 cells/ml in hypotonic buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM KCl, and 5 mM MgCl2 (TKM). Swelled cells were homogenized in a Dounce-type homogenizer until ~95% rupture was achieved, as
assayed by trypan blue staining. A post-mitochondrial supernatant was
prepared by centrifugation of the homogenate for 15 min at 10,000 × g. A microsome fraction was then prepared from the
post-mitochondrial supernatant by centrifugation for 15 min at
100,000 × g in a Beckman TLA 100.3 rotor. The
microsome fraction was resuspended in TKM containing 110 mM
KCl and 250 mM sucrose and solubilized by addition of
Nikkol to 1% (v:v) and deoxycholic acid to 0.5% (w:v). The solubilized membrane fraction was placed over a 1.5 M
sucrose cushion, and the ribosome fraction collected by centrifugation for 1 h at 80,000 rpm in a Beckman TLA 100.2 rotor. The ribosome pellet was resuspended in TKM containing 110 mM KCl. Rat
liver microsomes prepared by the method of Gaetani et al.
(13) were processed in the identical manner. Each ribosome preparation
was diluted to 1 A260 unit/ml in TKM and
supplemented with 2 mM octyl glucoside. A 15-µl drop of
each sample was placed atop a carbon-coated copper grid for 1 min at
room temperature. Grids were washed twice with TKM and stained with 2%
uranyl acetate for 10 s. Dry grids were viewed immediately after
drying or stored at room temperature.
Morphometry--
High magnification TEM images were recorded on
film with a microscope-mounted plate camera. The resulting negatives
were digitized with a CCD camera and imported into Adobe Photoshop
version 4.0. Digital images were adjusted for contrast and brightness
and then cropped to remove identification marks. Each image was
randomly assigned a code to reduce the possibility of operator bias
during subsequent analysis. Morphometric analysis of the images was
conducted with ObjectImage software (University of Amsterdam,
Netherlands). For the analysis, a transparent digital overlay was made
for each image. The overlay was then manually marked with segmented
line traces along transversely sectioned ER membrane bilayers.
Ribosomes intersecting the segmented lines were individually marked and counted as point particles, such that each line and its associated particles comprised one case. 697 cases and a total of 2170 ribosomes were measured from 37 micrographs representing the four experimental conditions. To compile the raw data, the micrographs were decoded, and
the cases were grouped by micrograph and by experimental condition. A
value representing the number of ribosomes per linear micron of
membrane was calculated for all cases in each micrograph. An average
value for the number of ribosomes per linear micron was then obtained
for each condition. For statistical analysis of the number of
membrane-bound ribosomes, analyses of variance and Student's
t tests were performed on data sets of size n for
each condition, where n was the number of micrographs.
p values were assigned to each of the data sets from
inhibitor-treated cells based on the T statistic, where
p Cell Fractionation--
BRL cells were fractionated by
sequential detergent extraction as follows. Cells at 70-80%
confluency were harvested by trypsin/EDTA treatment and centrifugation.
The cells were washed and resuspended in phosphate-buffered saline at
4 °C to a concentration of 2 × 106 cells/ml.
One-ml aliquots of cell suspension were centrifuged at 1000 × g for 3 min and resuspended in a cytosol buffer containing 110 mM KOAc, 25 mM K-HEPES, pH 7.5, 2.5 mM Mg(OAc)2, 1 mM EGTA, and 2 mM dithiothreitol. Digitonin was added to final
concentrations of 10-60 µg/ml. After 5 min of gentle agitation on
ice, the cell suspension was centrifuged at 1000 × g
for 3 min. The supernatant was removed, and the cell pellet was washed
twice in digitonin-supplemented buffer to yield a permeabilized cell
fraction that was enriched in membrane-bound polysomes and depleted of
free and cytoskeleton-associated polysomes. To release membrane-bound
polysomes, the extracted cell pellet was then resuspended in a buffer
containing 1% (v/v) Nikkol, 0.5% (w/v) deoxycholic acid, 110 mM KOAc, 25 mM K-HEPES, pH 7.5, and 5 mM Mg(OAc)2. After 2 min of gentle agitation
the cell suspension was centrifuged at 3000 × g for 5 min, and the supernatant fraction was collected.
Ribosome Detection--
To radiolabel ribosomes, growth media
for BRL cells at 35% confluency were replaced with fresh media
supplemented with 10 µCi/ml of 5,6-[3H]uridine, and the
cells were cultured in the presence of [3H]uridine for
18 h. Where indicated, cycloheximide, puromycin, or pactamycin was
added 10 min prior to processing. For determination of total ribosome
content in subcellular fractions, equivalent samples were placed atop a
cushion of 1.5 M sucrose in buffer containing 110 mM KOAc, 25 mM K-HEPES, pH 7.5, and 2.5 mM Mg(OAc)2. The samples were centrifuged for
1 h at 80,000 rpm in a Beckman TLA 100.2 rotor, and the
ribosome-enriched pellet fractions were resuspended in 5% SDS for
determination of associated radioactivity. For the resolution of
ribosomes by subunit distribution, 200 µl of the digitonin soluble
fraction containing free ribosomes or the Nikkol/DOC-soluble fraction
containing the membrane-bound ribosomes were loaded onto continuous
0.5-1.5 or 0.3-0.9 M sucrose gradients containing 400 mM KOAc, 50 mM Mg(OAc)2, and 25 mM K-HEPES, pH 7.5. The gradients were centrifuged for
3 h at 35,000 rpm (4 °C) in the Beckman SW41 rotor.
0.4-ml gradient fractions were collected manually by tube puncture and
drop collection. The [3H]uridine content in duplicates of
each fraction was determined in a liquid scintillation spectrometer.
Samples were counted twice within 24 h, and an average value for
[3H]uridine content was calculated for each sample.
Characterization of Experimental System--
To study the temporal
coupling of ribosome exchange and protein translation on the ER
membrane, tissue culture cells were treated with protein synthesis
inhibitors selective for either the initiation or elongation cycle of
protein translation, and ribosome-membrane interactions were assayed by
morphometric and biochemical methods. Three protein synthesis
inhibitors were investigated as follows: cycloheximide, puromycin, and
pactamycin. The mode of action, site of action, and the predicted
effects of each inhibitor on polysome structure are listed in Table
I.
Because tissue culture cell lines often display differences in their
sensitivity to protein synthesis inhibitors, each inhibitor was assayed
to identify effective concentrations and to screen for secondary
effects on protein secretion. In these experiments, methionine-starved
HepG2 cells were pulsed with [35S]methionine in the
presence or absence of inhibitor, and the rate of isotope incorporation
into albumin was determined by immunoprecipitation, SDS-PAGE, and
PhosphorImager analysis. As shown in Fig.
1A, in the absence of
inhibitor, [35S]methionine incorporation increased
linearly over the time course of the assay (7.5 min). At the
concentrations used, cycloheximide and puromycin rapidly and
effectively inhibited incorporation of
[35S]methionine into albumin. In the presence of
pactamycin, [35S]methionine incorporation into albumin
proceeded linearly for a short time and then ceased. For pactamycin,
the pattern of isotope incorporation was as predicted for an inhibitor
of initiation; only those nascent chains undergoing elongation at the
time of pactamycin addition were completed, and subsequent de
novo protein synthesis was blocked. An additional series of
controls was performed to identify any secondary effects of the protein
synthesis inhibitors on protein secretion. In these experiments,
pulse-chase studies of albumin synthesis and secretion were performed
in the presence of each of the inhibitors. The kinetics of albumin
secretion in the presence of either cycloheximide, puromycin, or
pactamycin were essentially identical, with a half-time for secretion
of approximately 20-30 min (Fig. 1B). In summary, the three
inhibitors used in this study disrupt the protein synthesis activity of
HepG2 cells in the manner predicted from their established mode of
action. Furthermore, these data demonstrate that the experimental
manipulations necessary to evaluate the fate of ribosomal subunits
arising from premature (puromycin) or natural (pactamycin) termination
do not adversely alter ER function or the activity of the protein
secretion machinery.
Ribosomes Remain Membrane-bound after Termination, Morphometric
Analysis--
To characterize any changes in ribosome-membrane
interaction that might accompany the termination of protein synthesis,
a series of ultrastructural and biochemical studies were conducted with
cultured cells. In experiments on rough ER ultrastructure, HepG2 and
BRL cell monolayers were treated with the described protein synthesis
inhibitors for 10 min. This time period is sufficient to allow complete
inhibition of protein synthesis and, in the case of puromycin and
pactamycin, the discharge of nascent chains from the ribosome. Samples
were then placed on ice and fixed with glutaraldehyde. Ultrathin
sections were prepared, stained, and viewed by electron microscopy. A
representative section from untreated HepG2 cells is illustrated in
Fig. 2A. In this micrograph an
abundant and extensive ER network can be readily distinguished from
cytosol by the relatively dark staining of the membrane-bound ribosomes and the granular, lumenal reticuloplasm. The ER cisternae are seen in
both transverse transversely sectioned and obliquely sectioned membrane
sheets. Two high magnification views of polyribosomes on the rough ER
(Fig. 2, B and C) illustrate the resolution
observed in oblique and transverse sections. In the oblique view, a
polysome is visible in the commonly observed rosette pattern (14). The spatial orientation of ribosomal subunits can also be discerned in
these panels, where large subunits are oriented toward the outside of
the polysome. Small subunits appear atop the large subunits and face
the polysome interior.
The morphological analysis of ribosome-membrane interactions following
translation inhibition was restricted to transverse views of the ER
membrane. Extensive sets of micrographs were prepared for cells that
had been treated with cycloheximide, pactamycin, and puromycin, as well
as untreated cells. Representative micrographs of the kind used in the
analysis are depicted in Fig.
3A. A total magnification of
approximately 100,000 afforded clear views of individual ribosomes
along the length of transversely sectioned ER in untreated cells (Fig.
3A). The ribosomes are clustered in groups of 3-5 along
some regions, which indicates the association of these ribosomes in
polysomes at the time of fixation. Although it was not possible to
consistently resolve individual subunits at this magnification, the
asymmetric positive staining of the membrane-bound particles suggests
that both large and small subunits are bound in untreated cells. A
similar distribution of ribosomes along the ER membrane was observed in
cells after treatment with cycloheximide (not shown).
Visual inspection of transverse ER sections from pactamycin- and
puromycin-treated BRL cells revealed an extensive number of
membrane-bound ribosomes, as shown in Fig. 3, B and
C. This finding was surprising in light of the activity of
the inhibitors and the predicted behavior of ribosomes upon
termination. Following run-off translation or premature termination,
the ribosomes would be expected to dissociate into subunits and
discharge from the membrane. However, we observed a distribution of
membrane-bound ribosomes which approximated that seen in untreated and
cycloheximide-treated cells, where the majority of membrane-bound
ribosomes contained translocating nascent chains. Along the plane of
the membrane, the ribosomes of pactamycin- and puromycin-treated cells
did not have the same degree of lateral organization found in control cells (Fig. 3A). A more random distribution of ribosomes in
pactamycin or puromycin-treated cells may be attributed to the loss of
polysome structure. Thus, following pactamycin or puromycin treatment, no rosette or linear polysome patterns in the cytoplasm or on the rough
ER membrane were observed (data not shown).
To evaluate quantitatively the distribution of bound ribosomes,
micrographs of cells from all experimental conditions were digitized
and adjusted to uniform contrast and brightness levels (see
"Experimental Procedures"). Because of the small diameter of
ribosomes relative to resin sections and the propensity of polysome-associated ribosomes to extend above and below the plane of
section, it is difficult to quantify the number of ribosomes present
per unit area of membrane in an oblique section (15, 16). However,
transverse sections in which the membrane-bound ribosomes are of
similar dimension can be quantitated and the relative ribosome density
expressed as ribosomes per linear micron. At least 430 individual data
points were collected for each treatment from 7 or more unique
micrographs. Differences between results from untreated cells and
inhibited cells were assessed using the Student's t test.
The results of the analysis are depicted in Table
II. Following elongation arrest with
cycloheximide, ribosomes were present at a density of 12.17 ribosomes
per linear micron of ER membrane. This number was not statistically
different from the density of ribosomes measured in untreated cells,
indicating that no net release or addition of ribosomes occurred upon
incubation with cycloheximide. Following treatment with puromycin, the
density of bound ribosomes present in cross-section was 8.24 ribosomes/µm, a decrease of 35% compared with control. A similar
decrease was observed for cells treated with pactamycin, where 8.03 ribosomes/µm were present. Statistical analysis of the data indicate
that these differences from control cells are significant, with
p values of 0.0001. It is apparent from these results that
following release of the nascent chain from the ribosome upon either
premature termination or run-off translation, approximately one-third
of the ribosomes completely detach from the ER, whereas the majority
remain in stable association.
Biochemical Fractionation of Free and Membrane-Bound
Ribosomes--
Morphometric analysis of the ribosome-membrane
association in intact cells indicated that ribosomes and/or ribosomal
subunits are found in association with the ER membrane following
inhibitor-elicited termination. However, in traditional thin sections,
it is difficult to distinguish unequivocally large and small ribosomal
subunits. To gain insight into the structural composition of the bound
ribosomal particles remaining upon termination, inhibitor-treated cells were fractionated and ribosomal subunit identity determined by velocity
sedimentation on sucrose gradients. A method was developed to allow
rapid separation of membrane-bound and free ribosomes in BRL cells,
based on earlier fractionation procedures with this cell line (17, 18).
Cell monolayers were cultured for 18 h with
[3H]uridine to radiolabel RNA. Semi-intact cells devoid
of cytoplasm were subsequently generated by digitonin-based
permeabilization in isotonic buffer. The digitonin-extracted cells were
then solubilized with nonionic detergent, to release membrane-bound
ribosomes, and centrifuged to remove insoluble components.
Membrane-derived ribosomes were resolved by centrifugation through
continuous sucrose gradients or discontinuous sucrose cushions.
The digitonin concentration was optimized to allow efficient removal of
cytosolic ribosomes without affecting ER membrane integrity. As shown
in Fig. 4, 40 µg/ml digitonin was the
minimum detergent concentration necessary to elicit selective plasma
membrane permeabilization. Under these conditions, cytoplasmic
radiolabeled RNA species were removed with two washes in
digitonin-supplemented isotonic buffer. The radiolabeled RNA remaining
in the cell was not susceptible to further extraction at 4 °C and
thus was operationally defined as membrane-associated. Integral ER
membrane proteins were observed to remain in quantitative association
with the cell pellet (data not shown).
Large Ribosomal Subunits Remain Bound after Termination--
The
digitonin-based cell fractionation procedure was used to determine
biochemically the state of ribosome assembly following the termination
of protein synthesis. In these experiments,
[3H]uridine-labeled cells were incubated with
inhibitor-supplemented media for 10 min at 37 °C. For each analysis,
2 × 106 cells were then processed to yield the
cytosol and membrane-bound ribosome fractions. Ribosomes present in
these extracts were separated by velocity sedimentation on sucrose
gradients, and fractions from the gradients were analyzed for
[3H]uridine content by liquid scintillation spectrometry.
A representative set of experiments is shown in Fig.
5A, which depicts ribosome migration profiles for the cytosol fraction of inhibitor-treated cells.
To identify the peaks in the upper portion of the gradient, the
positions of purified rat liver 60 S ribosomal subunits and 80 S
ribosomes were determined in parallel gradients (not shown). By
comparison with these standards, the three major
[3H]uridine-labeled peaks represent, respectively, 60 S,
80 S, and polysomal fractions. As predicted for the inhibitors used in
this study, cycloheximide-treated ribosomes maintain polysome
structure, whereas pactamycin- and puromycin-treated polysomes
dissociate into monomers and subunits. Monomer dissociation was
accompanied by a compensatory increase in the size of the 80 S and
60 S peaks (Fig. 5A). Small subunits were not well resolved
under these gradient conditions.
The migration profiles of the membrane-associated ribosome fraction
(Fig. 5, B and C) differ markedly from those of
the cytosolic fraction. Consistent with the findings from the
morphometric analysis, we observed that ribosomal subunits remained in
association with the ER membrane in the absence of nascent polypeptide
chains and polysomal structure. In the presence of pactamycin or
puromycin, the dominant radiolabeled, membrane-bound species is the
large ribosomal subunit. 40 S subunits were present only at
sub-stoichiometric levels (Fig. 5C). 80 S ribosomes are
nearly absent on the membrane, indicating that termination and
subsequent dissociation of the large and small subunits had occurred.
In contrast, the membrane-bound ribosomes from cycloheximide-treated
cells have not undergone termination and thus remain predominantly in
polysomes. These results indicate that after nascent chain discharge,
small ribosomal subunits are preferentially released from the ER
membrane, whereas large subunits remain bound.
Ultrastructural Analysis of Membrane-bound Ribosomal
Subunits--
Biochemical studies with permeabilized cells showed that
large subunits remain bound to the ER membrane after termination, whereas small subunits were preferentially released. To evaluate further these findings, a series of experiments was conducted to image
the membrane-associated particles after inhibitor treatment. BRL cells
were treated with cycloheximide, pactamycin, or puromycin, and a
microsomal fraction was prepared. After solubilization of the microsome
fraction and centrifugation, the soluble microsomal contents were
adsorbed onto copper grids, negatively stained with uranyl acetate, and
viewed by transmission electron microscopy. Images of membrane-derived
ribosomal subunits after experimental treatments were compared with
purified subunits, and polysomes from rat liver microsomes were
prepared in the same manner. Determination of ribosome structure after
negative staining was based on previously published results (19,
20).
Images of negatively stained small subunits, large subunits, and 80 S
ribosomes from rat liver are shown in Fig.
6. 40 S subunits (Fig. 6A)
are characterized by an elongated profile with one or two
stain-accessible regions that traverse the subunit and are sometimes
accompanied by indentations along the sides (see Ref. 19, plates I and
II). 60 S subunits (Fig. 6B) are generally round and may
also appear "skiff-shaped." Their most striking characteristic is
the appearance of one or more small protrusions on one side of the
subunit in the skiff orientation. In this view, the opposite side is
demarcated by an even deposit of stain around the periphery of the
subunit (see Ref. 19, plates III and IV). Monomeric ribosomes and
polysomes are noticeably larger than either 40 S or 60 S subunits
(Fig. 6C). They are identifiable by their distinct bilobal
appearance, wherein a deposit of stain is visible at the large
subunit-small subunit interface. The interface is visible along the
width of the structure or as a dense spot on one side (see Ref. 19,
plates IX and XI).
The composition of membrane-derived subunits from inhibitor-treated
cells was identified on the basis of these standards. Composites of
these subunits are shown in Fig. 7. As
expected, treatment with cycloheximide yields polysomes and 80 S
monosomes (Fig. 7A). Negatively stained intact polysomes
were not as numerous as we predicted from sucrose gradient
centrifugation; this is likely due to degradation during the
homogenization and isolation procedure. Ribosomes from
cycloheximide-treated BRL membranes appear very similar to those from
untreated rat liver microsomes. The monomers are asymmetric and contain
a stain-filled groove between large and small subunits in the frontal
orientation.
In contrast, negatively stained particles from pactamycin- and
puromycin-treated BRL cell membranes (Fig. 7, B and
C) strongly resemble purified rat liver large subunits. They
share common features with large subunits from both our preparation and
that described in Ref. 19, including a round appearance and small protuberances on one side marked by heavier stain deposits in the skiff
orientation. The particles are also similar in diameter to purified
60 S subunits and noticeably larger than 40 S subunits (Fig.
6A), while lacking the large spot of stain that demarcates intact monomers. We conclude that the distribution and structure of
these membrane-bound ribosomal particles after premature or natural
termination in vivo is consistent with our biochemical observations of large subunit retention and small subunit dissociation.
We report that the termination of protein synthesis on
membrane-bound ribosomes in vivo results in the enhanced
release of small ribosomal subunits from the membrane-bound pool; large
ribosomal subunits remain in stable association with the ER membrane.
Although these data do not support the proposal that the termination of protein synthesis on membrane-bound ribosomes yields the release of
large and small ribosomal subunits into the free, cytoplasmic pool,
they are consistent with prior data demonstrating that, in
vitro, the release of nascent chains from membrane ribosomes results in the enhanced exchange of small but not large ribosomal subunits (21).
From a historical perspective, the question of whether membrane-bound
ribosomes participate in a translation-dependent exchange with free ribosomes closely followed the observation that free, cytosolic ribosomal subunits associate and dissociate coincident with
the initiation and termination stages of translation (22, 23). Since it
had been established that ribosomes were segregated between free and
membrane-bound pools, studies were thus performed to determine whether
such ribosome pools were kinetically exchangeable (6, 9, 11, 21, 24).
Two experimental approaches were taken. In one approach, radioisotope
incorporation studies were performed to determine the rate of entry of
ribosomal subunits into the free and membrane-bound pools of intact
cells. In the study of Baglioni et al. (9), the primary
conclusion was that large ribosomal subunits bind to the ER membrane,
and polysome assembly occurs as small subunit initiation complexes
joined with membrane-bound large subunits to yield translationally
active 80 S ribosomes. This conclusion was disputed by Mechler and
Vassalli (6, 11, 24) in a detailed and rigorous series of
investigations. These authors concluded that the kinetics of isotope
incorporation into large ribosomal subunits of the free and bound
ribosomal pools was such that the formation of 80 S ribosomes must
occur in the free ribosome pool. Mechler and Vassalli (11) did report, however, that the kinetics of membrane dissociation of large and small
ribosomal subunits was different, with large ribosomal subunit release
lagging that of small subunit release. In contrast to the above
referenced studies, which were performed in intact cells, Borgese
et al. (21) examined ribosomal subunit exchange in
vitro and reported that following the release of nascent chains,
ribosomal subunit exchange was strictly limited to small subunits;
large subunits remained in stable association with the microsomal
membranes. Because it could not be unequivocally concluded that the
in vitro system faithfully executed the termination reaction
as seen in vivo, and it was known that free and
membrane-bound ribosomal subunits were structurally and metabolically
similar, conclusions regarding the physiological significance of an
inexchangeable large ribosomal subunit pool were appropriately
conservative (21).
Although the isotope incorporation studies provided valuable data
demonstrating that free and membrane-bound ribosomes existed in
metabolic equilibrium, the experimental approach is hampered by a
significant kinetic constraint. That is, the rates of ribosomal subunit
biosynthesis and nuclear export are such that isotope incorporation
rates must be followed for periods of hours (6, 9, 11). Protein
synthesis, however, occurs in the time frame of seconds to minutes, and
thus the relevant exchange kinetics differ by 1-2 orders of magnitude.
With this experimental limitation in mind, we re-examined the fate of
membrane-bound ribosomes following the termination of protein synthesis
through a combined morphometric and biochemical analysis of
ribosome-membrane interactions in intact cells. The results of our
studies best support the hypothesis that the termination of protein
synthesis on membrane-bound ribosomes results in the free exchange of
small ribosomal subunits, with the large ribosomal subunits remaining
in stable association with the ER membrane. These data thus confirm and
extend the conclusions obtained by Borgese et al. (21) and
demonstrate that in the intact cell, large ribosomal subunit release
from the ER membrane does not occur coincident with the termination of
protein synthesis.
What is the fate of membrane-bound large ribosomal subunits after
termination? Should such subunits be competent for protein translation,
it is likely that protein synthesis could be initiated on the
endoplasmic reticulum membrane. Assuming this to be true, it then
becomes necessary to determine whether membrane-bound ribosomes can
select mRNA substrates and thus whether membrane-bound ribosomes
can catalyze the synthesis of free, cytosolic proteins. Furthermore, as
membrane-bound ribosomes are thought to reside in intimate association
with the protein conducting channel component of the ER translocon, it
is equally important that the compartmental fate of such translation
products be determined. As it is known that membrane-bound large
ribosomal subunits exchange with the cytoplasmic pool, it is essential
that the mechanism of large subunit release be determined and the
factors governing this release process be identified. Insights into
these questions are presented in the accompanying manuscript (31).
We gratefully acknowledge Drs. M. Reedy and
J. Corless for access to electron microscopy equipment and supplies;
and E. Worniallo, C. Lucaveche, and T. Zheng for excellent technical
assistance. We thank M. Potter, R. Lerner, C. Rioja and other members
of the laboratory for stimulating discussions. Portions of this study were performed with the electron microscopy facility of the Duke University Comprehensive Cancer Center.
*
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.
Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.M004462200
The abbreviations used are:
ER, endoplasmic
reticulum;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
DMEM, Dulbecco's modified Eagle's medium;
PAGE, polyacrylamide
gel electrophoresis.
The Fate of Membrane-bound Ribosomes Following the
Termination of Protein Synthesis*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.05 indicates a statistically significant
difference between the data sets from inhibitor-treated and untreated cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The effects of protein synthesis inhibitors on protein translation and
polysome structure
View larger version (52K):
[in a new window]
Fig. 1.
Analysis of protein synthesis inhibitor
activity. A, protein synthesis. HepG2 cells were incubated
in methionine-free DMEM at 37 °C and subsequently pulse-labeled with
100 µCi of [35S]methionine for 1 min prior to addition
of each inhibitor. At the indicated time points, which represent the
time after inhibitor addition, cells were harvested, and radiolabeled
albumin was collected from the cell lysate by immunoprecipitation.
Albumin content in each sample was quantitated from SDS-PAGE gels by
PhosphorImager analysis. B, protein secretion. Cells were
starved of methionine and subsequently pulsed with 100 µCi of
[35S]methionine; the chase period was initiated by
replacing the labeling medium with serum-free DMEM supplemented with 2 mM methionine and the indicated inhibitors. At each time
point, the medium was removed, and cells were chilled, rinsed, and
solubilized. Albumin was immunoprecipitated from the cell lysate
(C) and chase medium (M) as described under
"Experimental Procedures." Final inhibitor concentrations were 200 µM cycloheximide, 200 µM puromycin, or 0.2 µM pactamycin.
View larger version (155K):
[in a new window]
Fig. 2.
Ultrastructural analysis of endoplasmic
reticulum and membrane-ribosome morphology. A, HepG2
cells cultured on plastic coverslips were fixed in 3% glutaraldehyde
and processed as described under "Experimental Procedures."
Ultrathin sections were viewed in a transmission electron microscope at
60 kV; a representative micrograph is shown. Bar, 200 nm.
B, oblique section of a rosette-shaped polyribosome bound to
the rough ER. Bar, 50 nm. C, transverse section
of a membrane-bound polyribosome. B and C, large
and small subunits can be distinguished. Bar, 50 nm.
View larger version (97K):
[in a new window]
Fig. 3.
High magnification micrographs of endoplasmic
reticulum-rich sections of inhibitor-treated cells. Cells were
incubated in DMEM alone (A), 0.2 µM pactamycin
(B), or 200 µM puromycin (C) for 15 min prior to fixation and processing for transmission electron
microscopy, as described under "Experimental Procedures."
Representative micrographs of transversely sectioned ER were selected.
Micrographs from a common series were used for each morphometric
analysis.
Morphometric analysis of ribosome-membrane interactions following
premature or natural termination
0.05 indicates a statistically
significant difference between data sets from untreated and
experimental populations.
View larger version (32K):
[in a new window]
Fig. 4.
Isolation of membrane-bound ribosomes from
tissue culture cells. BRL cells were labeled with
[3H]uridine and harvested at 75% confluency. Cells were
washed and resuspended to 2 × 106 cells/ml in
isotonic buffer and subsequently treated with digitonin at the
indicated concentration for 5 min at 4 °C. Soluble cellular material
was then removed following brief centrifugation, and the cell pellet
was subjected to a series of washes in detergent-supplemented isotonic
buffer. To recover the membrane-bound ribosomes, cell pellets remaining
after the final wash were solubilized in a buffer containing 1% Nikkol
and 0.5% deoxycholic acid, and remaining nuclei and/or nuclear
fragments were removed by centrifugation. Cytosolic ribosomes released
upon digitonin extraction, and membrane-bound ribosomes released upon
detergent solubilization were recovered by sedimentation through 1.5 M sucrose cushions, and ribosomal RNA was quantitated by
scintillation counting.
View larger version (23K):
[in a new window]
Fig. 5.
Ribosomal subunit structure following
premature or natural termination. HepG2 cells were labeled with
[3H]uridine for 18 h and at the time of the
experiment incubated in DMEM supplemented with 200 µM
cycloheximide, 200 µM puromycin, or 0.2 µM
pactamycin for 15 min at 37 °C. Cells were then harvested, and the
cytosolic and membrane-bound ribosome fractions were collected
following treatment with an extraction buffer containing 40 µg/ml
digitonin, as described in the legend to Fig. 4. Cytosol and
membrane-derived ribosome fractions obtained by digitonin treatment
were subsequently fractionated by velocity sedimentation on sucrose
gradients. The amount of [3H]uridine in each gradient
fraction was quantitated by scintillation counting. A,
polysome profile of digitonin-releasable cytosolic contents.
B, polysome profile of solubilized ER membrane fractions of
digitonin-treated cells. A and B, ribosomes were
separated on a 0.5-1.5 M sucrose gradient. C,
ribosomal subunit profile of solubilized ER membrane fractions of
digitonin-treated cells. Subunits were separated on 0.3-0.9
M sucrose gradients.
View larger version (122K):
[in a new window]
Fig. 6.
Negative stain imaging of small and large
ribosomal subunits and monomeric ribosomes. Rat liver rough
microsomes were prepared by homogenization and centrifugation according
to standard protocols. Microsomes were solubilized in 1% Nikkol, and
the soluble ribosomes were isolated by centrifugation through sucrose
cushions. Ribosomal subunits were separated by treatment with a
puromycin-high salt treatment followed by sucrose gradient
centrifugation. Preparations containing 40 S subunits, 60 S subunits,
and 80 S ribosomes were adsorbed onto carbon-coated copper grids
without fixation. The grids were stained with uranyl acetate before
viewing in a transmission electron microscope at 100,000-150,000-fold
magnification. Representative particles were selected from micrographs
of each fraction. A, purified 40 S subunits; B,
purified 60 S subunits; C, microsome-derived 80 S monomers
and polysomes (lower left).
View larger version (125K):
[in a new window]
Fig. 7.
Negative staining of membrane-bound subunits
after termination of protein synthesis. BRL cells were treated
with 200 µM cycloheximide (A), 200 µM puromycin (B), or 0.2 µM
pactamycin (C) for 15 min before harvesting. Cells were
washed, resuspended in hypotonic buffer, and ruptured with a
Dounce-type homogenizer. A rough microsome fraction was prepared by
centrifugation on a discontinuous sucrose gradient and solubilized in
nonionic detergent. The membrane-derived ribosomal particles were
adsorbed onto EM grids and processed as described in the legend to Fig.
6. Representative particles were selected from micrographs of each
fraction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Cell
Biology, P. O. 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
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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