From the Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506
Received for publication, July 19, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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Proteins synthesized by the rough endoplasmic
reticulum (RER) co-translationally cross the membrane through the pore
of a ribosome-bound translocon (RBT) complex. Although this pore is also permeable to small molecules, it is generally thought that barriers to their permeation prevent the cyclical process of protein translation from affecting the permeability of the RER. We tested this
hypothesis by culturing Chinese hamster ovary-S cells with inhibitors
of protein translation that affect the occupancy of RBTs by nascent
proteins and then permeabilizing the plasma membrane and measuring the
permeability of the RER to a small molecule, 4-methyl-umbelliferyl- In eukaryotes, secretory and most integral membrane proteins are
synthesized by a translationally active 80 S ribosome composed of 60 S
and 40 S subunits and bound to a translocon complex, a heteromeric assembly of proteins embedded in the membrane of the rough
endoplasmic reticulum (RER)1
(Fig. 1). Nascent proteins emerging from the exit tunnel of the 60 S
subunit co-translationally cross the membrane of the RER by passing
through a protein-conducting channel (PCC) in the translocon complex
(1). The exit tunnel of the 60 S subunit and the pore of the PCC must
be large enough to be permeated by a nascent protein chain, and this
pathway is therefore large enough to be permeated by many other small
molecules when a ribosome-bound translocon (RBT) is translationally
inactive and empty, i.e. the pore is not occupied by a
nascent protein. We previously demonstrated (2) that the permeability
of RBTs to a small, polar molecule (4-methyl-umbelliferyl- The permeation of translocons by small molecules has also been
investigated by Johnson and colleagues (7, 8), who concluded that a
permeability barrier is maintained at the luminal end of the translocon
by the binding of BiP, a luminal chaperone protein, and that the
junction between the 60 S subunit and the translocon is tightly sealed.
Their conclusions have led to the broadly held view that RBTs are
tightly sealed at all times, preventing the process of protein
translation from influencing the permeability of the RER to small
molecules. We propose, however, that the notion that the permeability
of the RER is not affected by permeation of RBTs might be limited to
experimental conditions in which translationally inactive, empty
ribosomes have been stripped from ER microsomes by a high-salt wash.
This experimental detail is important because it limits the detection
of permeation to only two states of the translocon, the translationally
active 80 S-bound translocon and the ribosome-free translocon
(blocked and closed states, respectively, in Fig.
1). A novel feature of our current study
is that we have used experimental conditions in which permeation of a
third state of the translocon, translationally inactive 60 S-bound
RBTs, can also be detected.
-D-glucopyranoside (4-M
G). The
premature or normal release of nascent proteins by puromycin or
pactamycin, respectively, increased the permeability of the RER to
4-M
G by 20-30%. In contrast, inhibition of elongation and the
release of nascent proteins by cycloheximide did not increase the
permeability, but it prevented the increase in permeability by
pactamycin. We conclude that the permeability of the RER is
coupled to protein translation by a simple gating mechanism whereby a
nascent protein blocks the pore of a RBT during translation, but after
release of the nascent protein the pore is permeable to small molecules as long as an empty ribosome remains bound to the translocon.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (4-M
G)) was
increased after puromycin, a tRNA analog, prematurely terminated
translation and released nascent protein chains from the PCC (path
a, Fig 1). The increased permeability to 4-M
G was
consistent with a previous report that RBTs incorporated into planar
bilayers and opened by puromycin are permeable to ions (3), and our
results were also supported by a recent report that puromycin can
release calcium from the RER (4). The permeation of empty RBTs
is especially significant in the context of recent studies by
Nicchitta and colleagues (5, 6), who reported that approximately
two-thirds of the 60 S subunits remain bound to translocons after the
normal completion of protein translation, thereby constituting a large pool of persistent, translationally inactive RBT complexes. Although permeation of these empty RBTs could have very important consequences for RER-dependent signaling and the maintenance of
essential gradients across the RER membrane, this pathway has received
relatively little attention.
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Fig. 1.
Permeation of ribosome-bound
translocons. Following the completion of translation, either the
80 S ribosome (path d) or the 40 S subunit (path
a) can be released from the PCC (dark gray). Only
the translationally inactive, 60 S-bound state of the PCC is permeable
to 4-M G. The pathway is blocked by a nascent protein in the 80 S-bound state and closed in the ribosome-free state (2). PUR and PAC,
but not CHX, shift the majority of 80 S-bound translocons to the empty,
60 S-bound state (5) (path a). The 60 S subunit can
be released by high salt (path c), thereby closing the pore
(2).
In this article we examine two models that describe the relationship
between the translational activity of RBTs and the permeability of the
RER to a small molecule, 4-MG. The first model is a "static permeability" model in which RBTs are never permeable to small molecules other than nascent proteins, and the permeability of the RER
is therefore independent of translational activity. The second model is
a "dynamic permeability" model in which the pore of an RBT is
"gated" closed when it is occupied by a nascent protein, but the
pore of translationally inactive, 60 S-bound translocons is open and
permeable to small molecules. This simple physical mechanism for
coupling the permeability of the RER to protein translation predicts a
dynamic relationship in which increasing the level of protein
translation and the occupancy of RBTs by nascent proteins should
decrease the permeability of the RER, whereas inhibiting the initiation
of protein translation and decreasing the occupancy of RBTs by nascent
proteins should increase the permeability of the RER.
The appropriateness of the static versus dynamic
permeability models can be tested by determining the extent to which
the permeability of the RER is dependent on the level of protein
translation and the occupancy of RBTs by nascent proteins. The static
permeability model predicts complete independence, whereas the dynamic
permeability model predicts that the permeability of the RER is
inversely related to the level of occupancy by nascent proteins during
protein translation. The goal of the current study was to test these
predictions by measuring the permeability of the RER to 4-MG while
manipulating protein translation with drugs that inhibit protein
translation by different mechanisms of action. Puromycin (PUR)
resembles aminoacyl-tRNA, and it is a substrate for the
peptidyltransferase of the 60S subunit, accepting a nascent peptide and
causing the premature release of incomplete nascent proteins (9).
Pactamycin (PAC) inhibits the initiation of protein translation by
preventing formation of the 43S initiation complex, but it permits the
normal elongation and release of full-length proteins whose synthesis
was initiated prior to addition of the drug (10, 11). Cycloheximide
blocks elongation and prevents the normal completion and release of
nascent proteins from RBTs (12). Cycloheximide stabilizes polyribosomes and the association of 80S ribosomes with translocons, whereas PUR and
PAC elicit rapid breakdown of polyribosomes, but leave the majority of
translationally inactive 60S subunits bound to the RER (5). We report
that the permeability of the RER was sensitive to the manipulation of
protein translation and changes in the occupancy of RBTs by nascent
proteins, and we conclude that the permeability of the RER is
dynamically coupled to protein translation.
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EXPERIMENTAL PROCEDURES |
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Materials--
4-MG, cycloheximide, and puromycin-HCl
were from Calbiochem. Pactamycin was a generous gift from The
Upjohn Co. All other reagents were from Sigma.
Cell Culture-- CHO-S cells (Invitrogen), a cell line derived from Chinese hamster ovary K1 cells and selected for growth in suspension, was used for most experiments. A cell line derived from HEK-293 cells for growth in suspension (HEK-293-F) was also obtained from Invitrogen. HepG2 and BHK-21 cells were obtained from the ATCC (Manassas, VA). CHO-K1 cells were a gift from Ed Levitan (University of Pittsburgh). CHO-S and HEK-293-F cells were grown in serum-free medium (CHO-SFII and 293SFII medium, respectively, from Invitrogen). CHO-K1 cells were grown in Ham's F12 medium containing 5% serum, and BHK-21 and HepG2 cells were grown in Dulbecco's modified Eagle's medium containing 10% serum. All cells were cultured at 37 °C, 8% CO2.
4MG Assay--
The 4-M
G assay was performed as described
by Heritage and Wonderlin (2). Briefly, CHO-S cells and HEK-293-F cells
were typically grown to a density of 0.5-1.0 × 106
cells/ml in T-75 flasks or in spinner flasks. For a 32-well assay (eight conditions in quadruplicate), 20 ml of cells was pelleted and
resuspended in 20 ml of K-G buffer (140 mM potassium
glutamate, 2.5 mM MgCl2, 10 mM
HEPES, pH 7.25) and gently permeabilized by N2 cavitation
(2 min, 80 p.s.i.). This procedure was modified for adherent
cells. BHK-21 and CHO-K1 cells were grown in T-75 flasks, harvested by
scraping, and then homogenized in K-G buffer by N2
cavitation. HepG2 cells were grown to confluence in 48-well plates, and
the medium was replaced with K-G buffer containing 0.08% digitonin
(100 µl/well) for 5 min to permeabilize the cells and then diluted
1:5 with K-G buffer. Nunc 48-well plates were loaded with 0.5 ml of
sample/well, the plate with solutions was prewarmed to 35 °C, and
4-M
G (20 µM from a 20 mM stock in
methanol) was added immediately before placing the plate into a
CytoFluor 4000 plate reader. The fluorescence was measured for 30 min
at 2-min intervals with 10 s mixing before each measurement. The center wavelengths/bandwidths of the excitation and emission filters were 360/40 and 460/40, respectively.
Data Analysis--
In CHO-S cells, 4-MG is activated when it
enters the lumen of the RER and it is hydrolyzed by
-glucosidase II.
The slope of the fluorescence versus time curve,
S(t), is proportional to the rate of
entry and activation of the dye at time t. The linear and
exponential contributions to S(t)
were fitted by Equation 1,
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(Eq. 1) |
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(Eq. 2) |
Calcium Measurements--
CHO-S cells were loaded with 10 µM Fluo-3-AM (30 min, 23 °C) and then washed
twice in Hanks' balanced salt solution. Fluorescence emission at 510 nm was measured with excitation at 485 nm in a CytoFluor 4000 plate
reader. Adherent BHK-21 cells grown in 10% Dulbecco's modified
Eagle's medium (8% CO2, 37 °C) were released by
trypsinization and gentle trituration and then loaded with 10 µM Mag-Fura-2-AM (60 min, 37 °C) in Dulbecco's
modified Eagle's medium minus serum. The cells were washed twice in
Hanks' balanced salt solution and loaded into a stirred cuvette in a
SPEX Fluorolog spectrometer. The ratio of fluorescence measured
at 510 nm with excitation alternating between 340 and 380 nm was
calculated at each time point without further calibration.
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RESULTS |
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Rationale for Testing the Permeability Models--
We have studied
the permeation of RBTs in CHO-S cells by
4-methyl-umbelliferyl--D-glucopyranoside (4-M
G), a
small, membrane-impermeant probe; it's entry into the RER can be
detected when it is hydrolyzed to a fluorescent product by luminal
-glucosidase II. We previously reported (2) that the activation of
4-M
G in intact CHO-S cells was negligible, but the selective rupture
of the plasma membrane by N2 cavitation enabled 4-M
G to
access the RER, and it revealed a substantial basal permeability of the
RER to 4-M
G (Pbasal). This permeability was
further increased to a level termed PPUR when
nascent chains were released by the addition of PUR (Fig. 2).
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The static permeability model predicts that
Pbasal should be insensitive to changes in the
occupancy of RBTs by nascent proteins produced by concomitant changes
in protein translation. This model was tested using a two-step
procedure. In the first step, cells were pretreated in culture with
PUR, PAC, or CHX to produce changes in the occupancy of RBTs. In the
second step, these cells were permeabilized, split into two samples,
and P was determined in each sample either without PUR
(Pbasal) or with PUR
(PPUR) added to the sample, respectively. The
occupancy of translationally active RBTs by nascent proteins after the
pretreatment period was estimated by calculating the
PUR-dependent increase in P,
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(Eq. 3) |
Given the importance of Pbasal and
PPUR in these tests, we first examined in
greater detail three features of Pbasal and
PPUR in this system: (1) the similarity of
Pbasal and
PPUR
measured in CHO-S cells to other cell lines, (2) the specificity of
PPUR as a measure of the occupancy of
translationally active RBTs by nascent chains, and (3) the contribution
of RBT-mediated permeation to Pbasal.
Similarities in Permeability (P) among Different Cell
Lines--
We examined the activation of 4-MG in the absence and
presence of PUR in CHO-S, CHO-K1, HEK-293-F, BHK-21, and HepG2 cells. We observed in all of these cell lines a substantial
Pbasal and a similar 30-40% increase in
P when puromycin was added to the assay (Fig.
2B). The similarity between CHO-S and HepG2 cells is
especially notable, because Seiser and Nicchitta (5) reported that
approximately two-thirds of the ribosomes remain bound to the RER
membrane in HepG2 cells following either the premature termination of
translation by PUR or the normal completion of translation with
pactamycin. We conclude from these similarities that the mechanisms
that regulate the permeation of RBTs are probably also similar among
these cell lines and that the high Pbasal and the
PPUR are not unusual features of CHO-S
cells. These results support our use of CHO-S cells in the
present study as a model system for investigating the permeability of
the RER.
PPUR Is a Measure of the Pool of Translationally
Active RBTs--
We previously concluded (2) that
PPUR was produced by the release of nascent
chains from translationally active RBTs on the basis of the generally
recognized mechanism of action of puromycin as a tRNA analog (9), the
similar sensitivity to high salt of
PPUR and
the binding of ribosomes to ER membranes (13), and the absence of
PPUR in the presence of detergent, which
demonstrated that
PPUR required the entry of
4-M
G into a membrane-bound compartment. In the current study we
wanted to use the size of
PPUR to track changes in the size of the pool of translationally active RBTs (i.e. those RBTs occupied by nascent proteins that could be
released by PUR) in response to treatments with inhibitors of protein
synthesis. The puromycin reaction catalyzed by the peptidyltransferase
is essential for the release of nascent proteins, and we performed the
following additional experiments to provide a more rigorous test of the
specific dependence of
PPUR on the puromycin reaction.
Anisomycin is an inhibitor of the peptidyltransferase and the puromycin
reaction (14). When added simultaneously with PUR, 50 µM
anisomycin completely inhibited PPUR, but it
did not inhibit
PPUR when it was added 10 min
after PUR, sufficient time for the irreversible release of nascent
chains by PUR (Fig. 3A). The failure of anisomycin to significantly inhibit
PPUR when added after PUR demonstrated that
the ability of anisomycin to inhibit
PPUR
when applied with PUR did not result from nonspecific effects, such as
blocking the pore of empty RBTs. We conclude from the complete
inhibition of
PPUR when anisomycin was
applied with PUR that the production of
PPUR
required an anisomycin-sensitive peptidyltransferase reaction.
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The puromycin reaction is maximum at pH 8.5-9.5, with a
pKa of 7.2-7.6 and a steep decrease in rate at
neutral pH and below (15, 16). We observed that
PPUR was inhibited when the buffer was
acidified from pH 7.2 to pH 6.6 before the addition of PUR, but
acidification to pH 6.6 15 min after the addition of PUR had no effect
(Fig. 3B). The temporal dependence of the inhibition by low
pH leads us to conclude, again, that the inhibitory effect was on the
puromycin reaction and not the permeability pathway.
We conclude that the production of PPUR is
entirely dependent on the puromycin reaction catalyzed by the
peptidyltransferase center, and it is therefore a valid measure of the
release of nascent proteins from translationally active RBTs. The
measurement of
PPUR provides an alternative
approach for determining the size of the pool of translationally active
RBTs that is much simpler than other methods (e.g.
radiolabeling nascent proteins). It is also insensitive to
translationally active ribosomes in the cytosol, because only the
release of nascent proteins from translationally active ribosomes that
are bound to the RER can increase the activation of 4-M
G. Although
PPUR provides only a relative measure of the translational activity of RBTs, that was sufficient for our current study in which we wanted to use
PPUR to track
changes in the size of the pool of translationally active RBTs in
response to inhibitors of protein synthesis.
The Contribution of RBTs to Pbasal--
We have
observed over a 2-year period that Pbasal can
slowly change in CHO-S cells over the course of several weeks or
months, with Pbasal ranging from 0.2 to 0.7. This variability led us to speculate that the entry of 4-MG via
pathways other than RBTs might contribute a labile background component
to Pbasal and cause us to underestimate the
importance of changes in Pbasal mediated specifically by permeation of RBTs. In the absence of drugs that can
specifically inhibit the permeation of RBTs, we have estimated the
component of Pbasal contributed by the
permeation of RBTs, relative to the background permeability contributed
by other, unidentified pathways, by examining the sensitivity of
Pbasal to high salt, which has been reported to
release up to 85% of the translationally inactive ribosomes from the
RER membrane in rat hepatocytes (path c, Fig. 1) (13).
Although high salt might also inhibit RBT-independent pathways for the
entry of 4-M
G, we have demonstrated previously (2) that both the
concentration of salt producing half-maximal block and the slope of the
salt concentration-inhibition curve were identical for
PPUR and the salt-sensitive component of
Pbasal. We surmised from the remarkably similar
sensitivities to high salt that both salt-sensitive components were
produced by the entry of 4-M
G through a common pathway. Cells were
split into two samples, with one sample broken open in high-salt (300 mM K) K-G buffer and the other sample broken open in normal
(140 mM K) K-G buffer. A plot of
Pbasal measured in high-salt versus
normal buffer is shown in Fig. 3C. The dotted line with unity slope indicates the relationship expected if
Pbasal was not affected by high salt, and it is
clear that Pbasal measured in high salt was
depressed relative to Pbasal measured in normal salt, consistent with our previous report that
Pbasal was sensitive to high salt (2). Most
importantly, a regression line fitted to these data had a slope of 0.94 (±0.10), insignificantly different from a value of 1. We conclude from
the parallel downward shift of the relationship that
Pbasal was decreased on average by
0.2 in
high-salt relative to normal-salt buffers regardless of the size of
Pbasal in normal salt. The data in Fig.
3C also indicate that the apparent lower limit of
Pbasal is 0.2, from which we can conclude that
in some samples of cells 4-M
G enters the RER exclusively through
empty RBTs. These data indicate that the size of the salt-sensitive
permeability to 4-M
G was surprisingly constant compared with the
high variability of the salt-insensitive component.
Effect of Puromycin--
The primary test of the static
permeability model was to determine whether pretreating cells with PUR
prior to permeabilization could reversibly increase
Pbasal. CHO-S cells were pretreated with 200 µM PUR for 15 min to terminate translation and release nascent proteins from RBTs, after which half of the cells were permeabilized and assayed. Pretreatment with PUR significantly increased Pbasal while substantially reducing
PPUR (Fig.
4A), as expected if all of the
available nascent proteins were released from translationally active
RBTs during the pretreatment period. If the increase in
Pbasal resulted from the release of nascent proteins and the opening of RBTs, it should be reversed by restarting translation and increasing the occupancy of RBTs by nascent proteins. The remaining control and PUR-treated cells were pelleted, washed, and
transferred to control medium for 30 min, at which time
Pbasal was not significantly different from the
control level (Fig. 4A), demonstrating that restarting
protein synthesis could reverse the increase in
Pbasal.
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Effect of Pactamycin--
To ensure that the increase in
Pbasal produced by PUR was not an artifact of
the premature release of truncated proteins by PUR, we also examined
pactamycin, which should release full-length proteins. A 1-h
pretreatment with PAC significantly increased Pbasal and eliminated
PPUR (Fig. 4B), as observed with
PUR pretreatment. Similar, significant increases in
Pbasal were observed following pretreatments
with PAC for 5, 30, 60, or 360 min, indicating that the increase in
Pbasal was both rapid and sustained (data not shown).
The increase in Pbasal was eliminated when control and PAC-treated cells were permeabilized in a buffer in which the potassium glutamate concentration was increased to 300 mM (Fig. 4B), which supports a role for empty RBTs because translationally inactive ribosomes can be stripped from the RER by a high-salt buffer (13). Also, unlike PUR, the acute application of PAC to permeabilized control cells did not increase Pbasal (data not shown), which is consistent with the ability of PUR to release nascent proteins directly versus the PAC requirement that synthesis be completed and nascent proteins be released prior to permeabilization.
The similar increase in Pbasal produced by
pretreating intact cells with either PAC or PUR can be accounted for by
the conversion of blocked RBTs to open RBTs. The increase in
Pbasal was balanced entirely by the loss of
PPUR, resulting in no net increase in PPUR (Fig. 4, A and B),
which was expected if the increase in Pbasal
depended solely on the conversion of blocked RBTs to open RBTs along
path a in Fig. 1. Also, the increase in
Pbasal produced by both PUR and PAC was about
70% of the
PPUR measured in control cells
(Fig. 4, A and B, where
PPUR reflects the size of the translationally active pool in control cells), which was remarkably consistent with the
previous report of a persistent binding of
65% of 60 S subunits
after the release of nascent proteins (5).
Effect of Cycloheximide--
In contrast to PUR and PAC, a 1-h
pretreatment with 200 µM cycloheximide (CHX) did not
increase Pbasal (Fig. 4C). A similar lack of effect was observed with 5- or 360-min pretreatments (data not
shown), indicating that the different effects of PAC and CHX were not
limited to a specific point in time. The failure of CHX to increase
Pbasal demonstrated that the increased
Pbasal produced by PUR or PAC was not a
nonspecific consequence of inhibiting protein translation
(e.g. clearing of proteins from the lumen of the RER). The
fact that PAC, but not PUR, requires elongation for the release of
nascent chains led us to predict that CHX would prevent the increase in
Pbasal by PAC, but CHX should have much less of
an effect on the increase in Pbasal by PUR.
Co-treatment with CHX completely prevented PAC from increasing
Pbasal (Fig. 4C), whereas CHX
decreased PPUR by only 20-30% (data not shown).
PUR-dependent Release of Calcium in Intact
Cells--
We also examined changes in the permeability of the ER to
Ca2+ to determine whether changes in the permeability to
4-MG could be generalized to Ca2+ and as a control to
ensure that changes in the permeability of the ER could be observed in
intact cells in which no cytosolic constituents that might regulate the
permeability of RBTs could be lost, in contrast to the 4-M
G assay
that requires permeabilization of the plasma membrane for the entry of
4-M
G. Intact CHO-S cells were loaded with the Ca2+
indicator Fluo-3 and treated in parallel with PUR and thapsigargin (TG), a selective inhibitor of the Ca-ATPase in the ER (17), with the
response to TG providing a positive control for a Ca2+
signal produced by the release of Ca2+ from the ER.
Thapsigargin (100 nM) and PUR produced similar, triphasic
changes in [Ca2+] (Fig. 5,
A and B), both beginning with a transient
increase. The increase in [Ca2+] elicited by PUR was not
a nonspecific effect of inhibiting protein synthesis, because it was
observed in cells in which protein synthesis was already inhibited by
pretreatment with CHX (Fig. 5A). We also used Mag-Fura-2 to
monitor more specifically changes in the permeability to
Ca2+. BHK-21 cells were loaded with Mag-Fura-2 under
conditions in which the [Ca2+] in the lumen of the ER
could be preferentially monitored (18). Although we could not detect
changes in luminal [Ca2+] with the addition of PUR to
intact BHK-21 cells (data not shown), PUR significantly increased the
rate of release of Ca2+ from the ER in intact cells when
re-uptake was inhibited by TG (Fig. 5C), evidence that the
permeability to Ca2+ was increased. Our observation that
PUR increased the permeability of the RER to calcium in intact cells is
consistent with a recent report that PUR depleted calcium in the RER of
dialyzed or permeabilized cells (4), and our observations in intact
cells demonstrated that the changes in the permeability to 4-M
G
produced by PUR did not result from the loss of potential regulatory
factors following permeabilization of the plasma membrane.
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DISCUSSION |
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We conclude from the experiments reported above that the permeability of the RER can be dynamically coupled to the level of protein translation by changes in the number of empty (i.e. open) versus blocked RBTs. Dynamic coupling could be produced by a simple mechanism requiring only the following features: (1) the pore of the PCC and the exit tunnel of the 60 S subunit are blocked by a nascent protein when the RBT is translationally active; (2) following the normal completion of translation and the release of a nascent protein, some empty ribosomes remain persistently bound to translocons; (3) the pore of empty RBTs is permeable to small molecules; and (4) the relative distribution of blocked versus empty translocons is influenced by the level of protein translation. The apparent high barrier to the permeation of translocons by small molecules, as described by Johnson and colleagues (Ref. 7; also reviewed in Ref. 8), is probably limited to experimental conditions in which permeable, translationally inactive ribosomes have been stripped from translocons by high salt, leaving only impermeable, translationally active RBTs and closed, ribosome-free translocons. This conclusion is supported by our current and previous observation (2) that Pbasal was significantly lower when measured in a high-salt buffer, presumably a result of the dissociation of ribosomes from translationally inactive RBTs.
At a macroscopic level, the functional consequences of the dynamic
coupling between Pbasal and protein translation
depend on the dynamic range over which Pbasal
can vary, and we expect that this range will be affected by the
properties of different permeant molecules. For 4-MG, the release of
all of the nascent chains by PUR or PAC increased
Pbasal by
30% (we have not yet examined the
level to which Pbasal can be decreased by
increasing translational activity and converting empty RBTs to blocked
RBTs). However, the presence of both salt-sensitive and
salt-insensitive components of Pbasal measured
with 4-M
G provided evidence that it could enter the RER through one
or more channels or carriers in addition to empty RBTs, which was not
surprising given the small size and simple structure of 4-M
G. The
increased background permeability resulting from the permeation of more
than one pathway by 4-M
G reduced the apparent size of the increase
in Pbasal that was produced by the release of
nascent proteins. For larger molecules that can enter the RER by
permeating empty RBTs but not via smaller channels or carriers, the
dynamic range could be significantly larger.
The dynamic coupling between Pbasal and protein
translation appears to occur in addition to a substantial contribution
of empty RBTs to the steady-state permeability of the RER. We measured the salt-sensitive component of Pbasal, which
includes, but is not necessarily limited to, the entry of 4-MG
through empty RBTs. From these measurements we estimated that
permeation of salt-sensitive, empty RBTs in the absence of puromycin
consistently contributed a fractional permeability to 4-M
G of
0.2 regardless of the size of Pbasal (Fig.
3C). Given an average size of
PPUR
of 0.10 (2), we estimate that the ratio of empty to occupied RBTs might
be as high as
2:1. This ratio is considerably higher than expected based on ultrastructural studies in which most ribosomes appear to be
part of translationally active polysomes (e.g. Fig. 174 in
Ref. 20). However, our results are more consistent with a study by
Adelman et al. (13) in which ~40% of the ribosomes could
be released from rat liver microsomes with salt alone, and an
additional 40-45% could be released with the combination of high salt
and puromycin. These authors concluded that the ribosomes released by
high salt in the absence of puromycin were mostly translationally
inactive, empty ribosomes, and their results would support a ratio of
empty to occupied RBTs of
1:1. Our results are also consistent with
a high permeability to small solutes observed in rough microsomes
prepared from rat liver without treatment with high salt (19).
Resolution of this issue will require parallel measurements of
permeability and ultrastructure in the same cells.
A high resting permeability to small molecules mediated by empty RBTs
also raises the question as to how the RER might maintain essential
gradients across its membrane or different environments (e.g. oxidizing versus reducing) in the lumen
versus the cytosol. Although the Ca-ATPase in the ER
membrane could maintain a calcium gradient in the presence of a leak
pathway, it is less clear which transporters might maintain gradients
for other molecules of interest. Of course, any speculation as to the
potential effect of empty RBTs on the permeability of the RER to small
molecules must be tempered by the fact that thus far we have only
demonstrated permeation by 4-MG and calcium, and further study will
be required to determine the repertoire of small molecules that can
permeate empty RBTs.
The macroscopic changes in Pbasal described
above might be especially important during global changes in protein
translation, such as when the initiation of protein synthesis is
inhibited in response to cellular stress (21). In this context, the
increase in Pbasal produced by PAC provides a
new clue into the mechanism of stress-induced apoptosis. Many cytosolic
and ER stressors inhibit the initiation of protein translation by
activating the phosphorylation of the translation initiation factor
eIF-2 (21), an effect mimicked by PAC. If inhibiting the initiation
of translation increases the permeability of the RER, as we observed
with PAC, then RER-dependent homeostatic processes might be
disrupted, thereby promoting apoptosis. The ability of cycloheximide to
stabilize nascent chains within RBTs and prevent PAC from increasing
Pbasal might explain why CHX can also prevent
stress-induced apoptosis under conditions in which protein synthesis is
already inhibited, such as during ischemia/reperfusion (22); and the
opposing actions of CHX and PUR on Pbasal might
account for the paradoxical, pro-apoptotic effects of PUR in situations
where CHX is protective (23, 24). From a methodological perspective,
experimental manipulations that stress cells and inhibit the initiation
of protein synthesis (e.g. removing extracellular calcium)
could have large effects on the permeability of the RER.
Dynamic fluctuations in Pbasal might also occur at a microscopic level as protein synthesis within local regions of the RER undergoes its normal cyclical course. For example, the permeation of empty RBTs by calcium or other small molecules following the release of a completed protein could produce a diffusible signal coupled to the end of translation, perhaps playing a role in stimulating the disassembly and reassembly of a translationally active RBT with successive cycles of protein translation. Molecules entering the lumen through an empty RBT might also signal its availability for the retrograde export of misfolded proteins through the pore of the RBT (25). At a microscopic level, the opening of RBTs might produce nearly quantal changes in Pbasal compared with the macroscopic changes in Pbasal averaged over larger dimensions. For example, the opening of an RBT could produce a rapid and large change in the local concentrations of molecules within the lumen of the RER, given the very small volume of the lumen and the potentially large flux of molecules through an empty RBT.
In conclusion, the permeability of the RER to small molecules should be
viewed as a dynamic property influenced by protein translation. We
propose that dynamic coupling could occur in any cells in which empty
ribosomes remain bound to translocons following the completion of
translation. Recognition of the dynamic nature of this relationship
provides new insight into the generation of novel signals and the
potential loss of homeostatic regulation under pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Durham, Jim Mahaney, and Peter Mathers for their comments on a draft of this manuscript and also Dorothy Heritage for performing the assays.
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FOOTNOTES |
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* This work was supported by American Heart Association Grant AHA 0051268B.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 304-293-3159;
Fax: 304-293-6854; E-mail: wonder@wvu.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M207295200
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ABBREVIATIONS |
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The abbreviations used are:
RER, rough
endoplasmic reticulum;
ER, endoplasmic reticulum;
PCC, protein-conducting channel;
RBT, ribosome-bound translocon;
4-MG, 4-methylumbelliferyl-
-D-glucopyranoside;
PUR, puromycin;
PAC, pactamycin;
CHX, cycloheximide;
TG, thapsigargin;
CHO-S, Chinese
hamster ovary-S cells;
S0, initial slope;
P, permeability;
Pbasal, puromycin-independent
permeability;
PPUR, permeability in the presence
of puromycin;
PPUR, increase of
PPUR above Pbasal.
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