Translocon Pores in the Endoplasmic Reticulum Are Permeable to a
Neutral, Polar Molecule*
Dorothy
Heritage and
William F.
Wonderlin
From the Department of Biochemistry and Molecular Pharmacology,
Robert C. Byrd Health Sciences Center, West Virginia University,
Morgantown, West Virginia 26506-9142
Received for publication, March 17, 2001
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ABSTRACT |
The pore of the translocon complex in the
endoplasmic reticulum (ER) is large enough to be permeated by small
molecules, but it is generally believed that permeation is prevented by
a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl
-D-glucopyranoside (4M
G),
a small, neutral dye molecule, cannot permeate an empty translocon pore
by measuring its activation by an ER resident
-glucosidase, which is
dependent on entry into the ER. The basal entry of dye into the ER of
broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores
of nascent polypeptides, creating additional empty pores. The basal and
puromycin-dependent entries of 4M
G were mediated by a
common, salt-sensitive pathway that was partially blocked by spermine.
A similar activation of 4M
G was observed in nystatin-perforated cells, indicating that the entry of 4M
G into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject
the hypothesis and conclude that a small, neutral molecule can permeate
the empty pore of a translocon complex, and we propose that
translationally inactive, ribosome-bound translocons could provide a
pathway for small molecules to cross the ER membrane.
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INTRODUCTION |
The endoplasmic reticulum
(ER)1 is the site of
essential synthetic and signaling processes, such as the synthesis of
secreted and membrane proteins and the production of calcium signals.
These processes require the transport of a broad spectrum of molecules across the ER membrane while maintaining a selectivity during transport
which prevents the loss of essential gradients. The ER membrane is rich
in pathways for the active and passive transport of molecules ranging
in size from ions and small polar molecules to proteins. The pore of
the translocon (sec61 complex) is the largest pore in the ER membrane,
with an estimated diameter of 40-60 Å in the ribosome-bound state and
a smaller diameter of 9-15 Å in the ribosome-free state (1, 2). When
bound by a ribosome, the pore of the translocon is aligned with the
peptide exit tunnel in the large subunit of the ribosome (3), and the average diameter of this tunnel is about 20 Å (4). Together, the
polypeptide exit tunnel of the ribosome and the pore of the translocon
provide a linked pore of sufficiently large dimensions to permit the
translocation of polypeptides across the ER membrane. The role of the
translocon pore as the pathway used for the cotranslational insertion
of nascent proteins into the ER is well established, and recent
evidence supports an even broader role for the translocon pore in
protein transport because the pore also provides a pathway for the
retrograde export of proteins from the lumen of the ER to the cytosol
(5, 6). A model for ribosome-translocon interactions in which the large
subunit of the ribosome remains bound to the translocon pore after
translation is terminated was proposed recently (7). An intriguing
implication of this model is that the ER membrane might contain a large
number of ribosome-bound translocon complexes with the pore unoccupied
by polypeptides.
The very large diameter of the pore of the ribosome-translocon complex
raises the possibility that many small molecules could permeate this
pathway when it is empty, i.e. unoccupied by polypeptides. According to the prevailing model, a large barrier to permeation of the
translocon pore is produced by the binding of BiP, a prominent ER
chaperone protein, to the luminal end of the pore (2, 8). This gate can
be opened only by nascent polypeptides that have grown to a length
greater than
70 amino acids. However, this model was developed using
charged molecules to probe the permeability of the translocon pore, and
the question remains whether the binding of BiP to the pore actually
produces a tight mechanical seal that blocks the passage of all
molecules, or, alternatively, does the binding of BiP produce a looser
seal that functions as a selectivity filter, allowing some molecules to
pass through? In particular, is there a barrier to permeation of the
translocon pore by neutral, polar molecules? The answer to this
question is important because permeation of the translocon pore by
small neutral molecules could play an essential role in bidirectional
signaling or the transport of substrates between the lumen of the ER
and the cytosol.
We have tested the hypothesis that the translocon pore maintains a high
barrier to permeation by neutral, as well as charged molecules by
measuring the entry into the ER of a small polar molecule,
4-methylumbelliferyl
-D-glucopyranoside (4M
G). The rationale for this assay was that entry of 4M
G into the ER can be
detected if it is cleaved by
-glucosidase II, a resident ER glucosidase (9-14), with the release of a fluorescent product. The
rate of accumulation of the fluorescent product should then provide a
measure of the rate of entry of 4M
G into the ER.
-Glucosidase II
has been purified from the ER (7, 9, 10, 12, 13, 15). It is a soluble,
heterodimeric enzyme that contains an HEDL ER retention signal (15),
and immunohistochemical staining has demonstrated that it is
specifically expressed in the lumen of the ER and some transitional
elements of the ER (14).
-Glucosidase II specifically cleaves
1,3-glycosidic linkages, and its activity is optimal at pH 6.5-7.0
(9, 10, 13, 15, 16), which clearly distinguishes it from the acidic
-glucosidase found in the lysosomes of some cells (17). The activity
of
-glucosidase II can also be distinguished from
-glucosidase I,
another neutral ER glucosidase (18, 19), by the fact that 4M
G is not
hydrolyzed by
-glucosidase I (19). We report that the empty
translocon pore is highly permeable to 4M
G, and we propose that
translationally inactive ribosome-translocon complexes might provide a
pathway for the movement of small, neutral molecules across the ER membrane.
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EXPERIMENTAL PROCEDURES |
Materials--
4M
G and puromycin HCl were from Calbiochem.
All other reagents were from Sigma.
Preparation of Permeabilized Cells--
CHO-S cells (Life
Technologies, Inc.) were grown to a density of 0.5-1.0 × 106 cells/ml in stirrer flasks in serum-free medium (Life
Technologies, Inc.) at 37 °C in 5% CO2. For a 32-well
assay (eight conditions in quadruplicate), 20 ml of cells was pelleted
and resuspended in 20 ml of KG buffer (140 mM potassium
glutamate, 2.5 mM MgCl2, 10 mM
HEPES, pH 7.25). The resuspended cells were pressurized for 2 min at
80 p.s.i. of N2 in a Parr nitrogen cavitation
homogenizer and gently broken open by release through the needle valve.
Under these conditions, 53% of the cells were permeabilized, as
measured by propidium iodide staining. Gentle opening of the plasma
membrane was essential. Increasing the N2 cavitation
pressure produced a disproportionate increase in the base-line
activation of 4M
G, a likely result of increased breakage of the ER
with the more vigorous cavitation. Permeabilization with 100 µM digitonin, which was 100% effective, increased the
variability of our measurements, perhaps as a result of destabilization
of the ER membrane by the detergent. We also observed that any attempt
to pellet the broken cells produced a greatly increased background fluorescence.
Fluorescence Assay--
Assays were performed using Nunc 48-well
plates read in a CytoFluor 4000 (PE Biosystems) plate reader. 0.5 ml of
a suspension of broken cells was loaded per well. A stock solution of
20 mM 4M
G was prepared in methanol and diluted into KG
buffer at a final concentration of 20 µM, unless
otherwise noted. The center wavelength and bandwidth of the excitation
and emission filters were 360/40 and 460/40, respectively. All
measurements were made at 35-37 °C. The plate with solutions was
prewarmed to 37 °C for 15 min, and the dye was added immediately
before transferring the plate to the reader. The fluorescence was
measured for 30 min at 2-min intervals, with 10 s of mixing before
each measurement. Puromycin HCl was prepared as a 10 mM
stock solution in water and used at a final concentration of 100 µM.
Data Analysis--
The substrate 4M
G is nonfluorescent until
the glycosidic linkage between the glucose and coumarin dye moieties is
cleaved by
-glucosidase, releasing the free, fluorescent dye. The
activation of 4M
G is irreversible, and the slope of the fluorescence
versus time curve, S(t),
is proportional to the rate of activation of the dye at time
t. Under most conditions,
S(t) was a constant, but under some
conditions there was a gradual increase or decrease in
S(t) which followed a simple
exponential time course. The linear and exponential contributions to
S(t) were well fitted by Equation 1
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(Eq. 1)
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where S0 is the initial slope
(
F min
1)and k is an
exponential rate constant (min
1). Best fit
estimates of S0 and k for each well
were obtained using the Solver nonlinear curve-fitting routine in Excel
(Microsoft). Parameters for Michaelis-Menten and Hill functions were
estimated using the Levenberg-Marquardt nonlinear curve-fitting routine in Origin, version 6.0 (Microcal). Statistical analysis was performed using JMPin (SAS Institute). Averages are plotted ± S.E. of the mean.
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RESULTS |
Activation of 4M
G in Detergent-permeabilized Cells--
We
first examined the activation of 4M
G in a broken cell preparation
nonspecifically permeabilized by the addition of 0.05% sodium
deoxycholate. Representative data and fitted curves calculated from
Equation 1 are shown in Fig.
1A. The increase in
fluorescence over time was fitted well by a combination of linear and
exponential components, with the linear component dominating at
concentrations of 4M
G above 10 µM. At lower
concentrations of 4M
G, an exponential decay of the initial slope,
S0, was apparent, probably as a result of
depletion of the 4M
G substrate with time. There was no activation of
dye when 4M
G was added to KG buffer in the absence of cells (data
not shown). The dependence of S0 on the
concentration of 4M
G is shown in Fig. 1B. At 4M
G
concentrations below 100 µM, S0
values were fitted by a single Michaelis-Menten function with a
Km of
5 µM, which was close to the
Km of
-glucosidase II for 4M
G reported in
bovine mammary gland (10). Although the
-glucosidase activity at low
substrate concentrations could be characterized by a single kinetically
defined process, a second, highly variable component became
increasingly evident at concentrations of 4M
G
100 µM (Fig. 1B). We did not identify the source
of this variable component. We chose 20 µM as a standard working concentration for our experiments because it was below the
concentration at which the rate of activation became highly variable,
yet it was high enough to drive a sufficient steady-state influx into a
membrane-bound compartment (described below).

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Fig. 1.
Concentration-dependent
activation of 4M G. Panel A,
plot of fluorescence versus time. Each concentration of
4M G was replicated in four wells, and the fluorescence of each well
was measured at 2-min intervals. Each symbol is the average
fluorescence of replicate wells at each time point, and the solid
lines are curves calculated from Equation 1 using the average of
the S0 and k values fitted to a set
of replicate wells. The fluorescence at zero time has been subtracted
from subsequent points. Panel B, S0
plotted versus 4M G concentration for five data sets. The
S0 values for each data set have been normalized
to the S0 observed at 25 µM 4M G
to emphasize the similar affinity across data sets. The solid
line is a Michaelis-Menten function calculated using a
Km of 5.3 µM, which was estimated by
simultaneously fitting all of the data sets using a shared
Km but independent S0,max
parameters across data sets. The increased variability of the data
plotted as open circles is evidence of the variable, lower
affinity component (higher concentrations not shown), and these data
were excluded from the curve fitting.
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We next examined the rate of activation of 4M
G as a function of pH
to determine if 4M
G could be activated in CHO-S cells by an acidic
-glucosidase that is present in the lysosomes of some cells. The
activation of 4M
G was strongly dependent on pH, and it was clearly
attributable to glucosidase activity that was optimal between pH 6.2 and 7.5. There was no evidence of an acidic
-glucosidase (Fig.
2A), which has a pH optimum of
4.5 (17). We also performed an assay for
-glucosidase, which, if
present in CHO-S cells, might nonspecifically activate some 4M
G.
Equimolar substitution of 4M
G by 4-methylumbelliferyl
-D-glucopyranoside, the
anomer of 4M
G and
substrate for
-glucosidase, yielded no fluorescent product (Fig.
2B). This assay was performed under conditions in which the
activity of
-glucosidase added to the preparation was readily
detectable (Fig. 2B). We conclude that neither acidic
-glucosidase nor
-glucosidase is present in CHO-S cells, and
4M
G is activated in CHO-S cells by
-glucosidase II.

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Fig. 2.
Panel A, pH dependence of
-glucosidase activity. Detergent-permeabilized cells in solutions
buffered to pH values between 4.7 and 7.5 were incubated for 30 min
with 10 µM 4M G, after which 100 µl of a 2.5 M Tris stock solution (pH 9.0) was added to bring the pH of
all of the test solutions to the same final pH. Tris is also an
inhibitor of -glucosidase II (10), and it effectively stopped the
activation of 4M G. Alkalization of the buffer prevented the
pH-dependent fluorescence of the activated dye
(pKa 8.0) from masking differences resulting
from pH-dependent glucosidase activity. For each of three
data sets, the S0 values are expressed as a
percentage of the S0 at pH 7.5, and the average
values are plotted versus pH. Panel B,
-glucosidase is not present in CHO-S cells. Experiments were
performed using 10 µM 4M G or 4M G substrate. 4M G,
but not 4M G, was activated in the presence of
detergent-permeabilized CHO-S cells. Positive controls consisting of
purified -glucosidase and -glucosidase, each at 0.05 unit
ml 1, demonstrated the equivalent sensitivity
of our assay to both glucosidases.
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Activation of 4M
G in Cavitation-permeabilized CHO-S
Cells--
The addition of 4M
G to the solution bathing intact CHO-S
cells resulted in very little activation of the dye (Fig.
3A), indicating that 4M
G
does not easily cross intact membranes. To enable access of 4M
G to
the ER, we chose low pressure N2 cavitation as a gentle method to break open the plasma membrane. The addition of 4M
G to
cavitation-permeabilized cells produced a surprisingly high basal rate
of activation, S0,basal, ~20-45%
(median = 28%) of the rate observed in cells permeabilized by
0.05% sodium deoxycholate (Fig. 3B). This high basal rate
was especially remarkable given that cavitation permeabilized only
slightly more than 50% of the cells, whereas the detergent
permeabilized all of the cells (data not shown).

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Fig. 3.
Activation of 4M G in
cavitation-permeabilized cells. Panel A, basal and
puromycin-dependent activation of 4M G in a
representative experiment. Intact cells diluted to the usual
concentration in KG buffer activated 4M G at a low rate.
Cavitation-permeabilized cells produced a substantial basal activation
of 4M G (S0,basal), which was increased
further by the addition of 100 µM puromycin
( S0,pur). The addition of detergent (0.05%
sodium deoxycholate) further increased the rate of activation.
Expression of the S0 measured in the absence of
detergent as a percentage of the S0 measured in
a detergent-permeabilized control, as shown on the right
axis, facilitated our comparison of data across experiments and
helped control for the direct effects of some treatments on the
absorbance of light or -glucosidase activity. Panel B,
distributions of S0,basal and
S0,pur measured as shown in panel
A for 193 assays. Panel C, intact membranes protect
-glucosidase activity from trypsin. The circles are
fluorescence versus time plots for the activation of 4M G
in cells permeabilized by 0.05% sodium deoxycholate, either in the
absence (filled circles) or presence (open
circles) of 0.25% trypsin. The decreasing slope of
S0 as a function of time most likely results
from the gradual destruction of the -glucosidase. The
triangles are plots of the activation of 4M G in
cavitation-permeabilized cells without detergent, either in the absence
(filled triangles) or presence (open triangles)
of trypsin. Average estimates of k calculated for four
replicate wells in each combination of conditions are plotted in the
inset. Trypsin clearly produced opposite effects on
k in the absence and presence of detergent. Panel
D, regression of S0,basal on
S0,pur. The correlation coefficient was 0.52, and the y intercept was 17.7%.
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Although a high basal rate of entry of 4M
G into the ER might be
inferred from the high rate of activation, an alternate explanation is
that 4M
G was activated by
-glucosidase exposed to the buffer by
accidental breakage of the ER. We tested this with a protease protection assay using trypsin. When trypsin (0.25% w/v) was added to
detergent-permeabilized cells, S(t)
decreased exponentially with time (Fig. 3C), demonstrating
that
-glucosidase not protected by intact membranes could be
inactivated irreversibly by trypsin. In contrast, the same
concentration of trypsin added to cavitation-permeabilized cells in the
absence of detergent produced an exponential increase, rather than a
decrease, in S(t) (Fig.
3C). The lack of a time-dependent decrease of
S(t) in the absence of detergent demonstrated that the
-glucosidase was protected from proteolysis by
trypsin, from which we conclude that the basal activation of 4M
G was
produced by
-glucosidase sequestered inside a membrane-bound compartment.
Puromycin is an aminoacyl tRNA analog that terminates the elongation of
polypeptide chains, releasing nascent polypeptides from ribosomes and
clearing the proteins from the pore of the translocon (20, 21).
Puromycin-treated translocons are permeable to ions (22), and we tested
the prediction that translocon pores cleared of protein by puromycin
would provide additional open pores through which 4M
G could enter
the ER. The addition of 100 µM puromycin increased
S0 to a value, S0,pur,
which was significantly greater than the basal rate (Fig.
3A). The increase in S0 produced by
the addition of puromycin,
S0,pur = S0,pur
S0,basal, was about 30% greater than S0,basal. We tested
puromycin at concentrations between 100 and 500 µM, and
we observed maximal dye entry at 100 µM puromycin (data
not shown). We conclude from the high specificity of puromycin for
terminating translation and the fact that the ER is the only
membrane-bound compartment to which ribosomes are attached that the
puromycin-dependent activation of 4M
G was produced by
the entry of 4M
G into the ER through ribosome-bound translocons.
The source of the puromycin-independent, basal activation of 4M
G was
less clear. As a first step, we tested by correlation analysis the
hypothesis that
S0,pur and
S0,basal were independent processes. There was a
significant correlation (r = 0.49, p < 0.001) between
S0,pur and
S0,basal (both calculated as a percentage of a
detergent control) across a large number of assays (Fig. 3D,
n = 142), and we reject the hypothesis of independence.
We estimated the portion of S0,basal which could
not be accounted for by variability in
S0,pur
by regressing S0,basal onto
S0,pur, and this uncorrelated residual was
~36% of S0,basal or 18% of the detergent
control (Fig. 3D). The correlation in the large data set was
probably reduced by variability in the magnitude of
S0,basal and
S0,pur
over months of experiments, and the correlation between
S0,pur and S0,basal
was substantially higher (reaching values of 0.75) when smaller data
sets (n < 20) collected over shorter periods of time
were analyzed (data not shown). The correlation and regression analysis
demonstrated that the processes underlying S0,basal and
S0,pur
were not independent, and a significant portion of
S0,basal could be predicted from
S0,pur.
Dependence on the Concentration of 4M
G--
The activation of
4M
G is irreversible, and the hydrolysis of 4M
G by
-glucosidase
could deplete the 4M
G concentration within a membrane-bound
compartment if it is not replenished by a continuous entry of dye. The
rate of entry of 4M
G is dependent on the permeability of the
membrane to 4M
G and the concentration gradient for 4M
G across the
membrane. This should produce an increase in the apparent Km of the
-glucosidase for 4M
G, with the
magnitude of the increase inversely proportional to the permeability of the membrane to 4M
G. We measured the dependence of
S0,basal and
S0,pur on
the concentration of 4M
G in cavitation-permeabilized cells to test
this prediction. Both sets of data were fitted by Michaelis-Menten
functions (Fig. 4). The
S0,pur data were fitted by a single component
with an apparent Km near 80 µM (Table
I). The S0,basal
data were fitted with two components, a dominant component with an
apparent Km also near 80 µM and a very
small, high affinity component. The apparent Km of
S0,pur and the dominant component of
S0,basal were increased ~16-fold relative to
the Km of the enzyme with detergent-permeabilized membranes (i.e. 5 µM, Fig. 1), and we conclude
from the very similar shift in the apparent Km
values that S0,basal and
S0,pur are produced by the entry of dye via
pathways with very similar permeabilities to 4M
G. The small, high
affinity component in S0,basal might represent
the activity of
-glucosidase released into the buffer, but the very
small size of this component (<3% of total) provides additional
evidence that nearly all of the activation of 4M
G in
cavitation-permeabilized cells occurred within a membrane-bound
compartment.

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Fig. 4.
Shift in the apparent
Km with intact membranes. Average
values for S0,basal (triangles) and
S0,pur (circles) are plotted
versus 4M G concentration (data pooled from 19 assays).
Each data set was fitted with a Michaelis-Menten function, and the
curves were calculated using the best fit parameters given in Table I.
The apparent Km for both data sets was near 80 µM, a 16-fold rightward shift compared with the curve
observed when membranes were permeabilized by detergent. The
dotted line is the fitted curve shown in Fig. 1B,
rescaled to the S0,max for the
S0,basal data set. Data at high concentrations
were variable, and the averages plotted as open symbols were
excluded from the fits. The methanol concentration at 10 µM 4M G was 0.05%. Addition of up to 5% methanol
(control for 1,000 µM 4M G) had no effect on the
activation of 10 µM dye (data not shown).
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Table I
Membrane-dependent shift in apparent Km
The dependence of S0 on 4M G concentration was
characterized by fitting the data sets with the function
S0 = S0,max/(1 + Km/C), where S0,max is
the maximum slope, C is the concentration of 4M G, and
Km is the concentration of 4M G which produces
one-half S0,max. Parameter values are given ± S.E. of the parameter estimate.
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Effect of Ionic Strength--
The puromycin-dependent
activation of 4M
G is most easily accounted for by a
puromycin-induced release of nascent polypeptide chains from the pores
of translationally active translocons, with the empty pores providing
additional pathways for the entry of 4M
G. Ribosomes can be
dissociated from translocons by first releasing nascent polypeptide
chains with puromycin, then increasing the ionic strength of the buffer
above about 100 mM (23). Treatment with high salt has been
shown to eliminate the permeation of translocon pores by ions (22). We
examined the rate of activation of 4M
G in cavitation-permeabilized
cells broken open in KG buffer containing 50, 140, 200, or 300 mM potassium glutamate. Increasing the potassium glutamate
concentration decreased both S0,basal and
S0,pur in an identical,
concentration-dependent manner (Fig.
5). The
concentration-dependent decreases in S0,basal
and
S0,pur were fitted with a Hill function with very
similar parameter values (Fig. 5 and Table
II), a concentration midpoint near
150 mM and a Hill coefficient of 7.6. The high
cooperativity was consistent with the breakage of multiple salt bonds,
and the absence of a second component, as evident in the quality of the single component fit, indicated that all of the salt bonds had a
similar sensitivity to the salt concentration. The concentration midpoint we observed was appropriate for the salt-dependent
release of ribosomes (23). We conclude that the salt-sensitive step for
S0,pur is the salt-dependent
release of ribosomes from translationally inactive, unoccupied
translocons.

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Fig. 5.
High salt inhibits
S0,pur and
S0,basal. CHO-S cells were
permeabilized in solutions containing 50, 140, 200, or 300 mM potassium glutamate. Average values for
S0,basal (triangles) and
S0,pur (circles), expressed as a
percentage of a detergent-treated control, are plotted
versus potassium glutamate concentration for a total of six
experiments. Each data set was fitted with a Hill function, and the
curves were calculated using the best fit parameters given in Table II.
Increasing the salt concentration inhibited
S0,basal and S0,pur in
a parallel manner, with nearly identical values of the h and
Ki parameters.
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Table II
Salt-dependent inhibition
The slope data were normalized as a percentage of the detergent control
and fitted with a Hill function of the following form:
S(C) = Smax/[1 + (C/Ki)h] + Const.,
where Smax is the maximum relative slope,
C is the potassium glutamate concentration,
S(C) is the slope at C,
Ki is the concentration at which there is
half-maximal inhibition, h is the Hill coefficient, and
Const. is the salt-insensitive activity.
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Modeling the Salt-dependent Entry of 4M
G--
We
surmised from the striking similarities in the inhibition of
S0,pur and S0,basal by
high salt that the salt-sensitive release of ribosomes from unoccupied
translocons was also the most likely mechanism underlying the reduction
in S0,basal by high salt. This interpretation is
especially attractive given recent evidence that the majority of large
ribosomal subunits remain bound to the ER after the normal termination
of protein synthesis (24, 25), and this state would be equivalent to the ribosome-bound, unoccupied state of the translocon produced by
treatment with puromycin (25).
To strengthen this interpretation, we developed a quantitative model of
the salt-sensitive and salt-insensitive components of
S0 in the absence
(S0,basal) and the presence
(S0,pur) of puromycin (Fig.
6 and Appendix). This model has three
free parameters: f, the fraction of pores open in the
absence of puromycin; x, the permeability of a blocked pore
relative to an empty pore; and s, the fraction of empty
pores from which ribosomes can be removed by high salt. We measured
S0,basal and S0,pur in a
normal salt buffer (140 mM KG) and a high salt buffer (300 mM KG) (n = 14 experiments). The
salt-sensitive component of S0,pur was then calculated and entered into the model, and the parameters f,
x, and s were adjusted to optimize the fit to
S0,basal and S0,pur under
the normal and high salt conditions. As shown in Fig. 6B, the fit was very good with parameter estimates of f = 0.738, x = 0.106, and s = 0.775. These
parameter estimates were robust and independent of the starting values.
Although this analysis indicated that 4M
G might permeate the blocked
pores to a limited extent, it is clear from Fig. 6B that
permeation of empty pores is the primary pathway. We conclude from this
analysis that the majority (74%) of the pores are permeable to 4M
G
in the absence of puromycin, and this result is consistent with the
persistent binding of ribosomes to translocons after termination of
translation (24, 25). We also note that the model predicted that 78%
of the unoccupied pores are sensitive to high salt, which is consistent with the observation by Adelman et al. (23) that a maximum
of
85% of the ribosomes bound to the ER can be removed by high salt after treatment with puromycin. Although our analysis does not rule out
all alternative models, it does demonstrate that a simple model based
only on the observed salt-sensitive entry of dye and three parameters
defining the puromycin- and salt-dependent regulation of
ribosome-bound translocon pores can reproduce exactly the distribution of S0,basal and S0,pur
under normal salt and high salt conditions.

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Fig. 6.
Panel A, model for
ribosome-translocon-dependent activation of 4M G.
Ribosome and translocon pores are depicted in three states:
left, the blocked pore of translationally active translocons
occupied by a nascent polypeptide; center, the empty pore;
and right, the closed pore produced by release of the
ribosome by high salt. The encircled B represents BiP bound
near the luminal opening of the translocon pore. The permeability of
the blocked state to 4M G is reduced greatly relative to the
permeability of the empty state to 4M G. All of the blocked pores and
a fraction (1 s) of the empty pores are insensitive
to high salt. The empty state can be induced by terminating translation
with puromycin, and it can also occur spontaneously. Panel
B, comparison of predicted and observed values of
S0,basal and S0,pur.
Cells were broken by cavitation and assayed in pairs, with one-half of
the sample of cells broken in 140 mM KG buffer and the
other half of the sample broken in 300 mM KG.
S0,basal and S0,pur were
calculated for each experiment (n = 14) as described in
the Appendix. The stacked bars show the contribution of each
component to S0,basal and
S0,pur. The salt-insensitive component shown
here was smaller than the value shown in Fig. 5. The salt-insensitive
component was generally more variable than the salt-sensitive
component, as evident in a 3-fold larger coefficient of variation (data
not shown).
|
|
Inhibition by Spermine--
If
S0,pur
and the salt-sensitive component of S0,basal are
produced by the entry of 4M
G through the same pathway, then agents that inhibit
S0,pur should also inhibit the
salt-sensitive component of S0,basal. We
predicted that spermine might block the pore of the ribosome-translocon
complex because it is a polyvalent cation of small size. 1 mM spermine inhibited
S0,pur by
70-90% while producing a smaller inhibition of
S0,basal (Fig.
7A). If the spermine-sensitive and the salt-sensitive components of S0,basal
represent the entry of 4M
G through the same pathway, then
elimination of the salt-sensitive component of
S0,basal by high salt should occlude the
inhibitory effect of spermine, and this was also observed (Fig.
7A). We also note that the apparent disparity in the
sensitivity of S0,basal and
S0,pur to spermine is much smaller if the
fractional block of the salt-sensitive components of
S0,basal and S0,pur are
compared, which were inhibited by 25 and 50%, respectively. The
inhibition of both S0,pur and
S0,basal by spermine provides additional support for our conclusion that they represent the entry of 4M
G via a common
pathway.

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|
Fig. 7.
Panel A, inhibition of
S0,pur and S0,basal by
spermine. 1 mM spermine in 140 mM KG buffer
reduced S0,pur to 20% of the control
response and produced a smaller inhibition of
S0,basal. In 300 mM KG buffer, both
S0,pur and S0,basal
were reduced greatly, and the inhibitory effect of spermine was
occluded. The averages for six experiments are plotted. Panel
B, activation of 4M G in nystatin-perforated cells. The
left set of bars shows a significant increase
(asterisks, p < 0.05, Tukey-Kramer honestly
significant difference test) in S0 with
the addition of nystatin and puromycin. The averages for nine
experiments are plotted. The relative sizes of the basal and
puromycin-stimulated activation of 4M G in nystatin-perforated cells
closely paralleled the proportions of S0,basal
and S0,pur observed in cells broken by
cavitation, although reduced in size by ~60-80%. The
right set of bars is the averages of three
experiments that tested the effect of spermine. 1 mM
spermine did not inhibit the activation of 4M G in
nystatin-perforated cells.
|
|
Activation of 4M
G in Nystatin-perforated Cells--
The
high rate of entry of 4M
G into the ER of cells broken open by
cavitation in the simple KG buffer might be explained by the loss of a
component of the cytosol which is required to maintain a permeability
barrier. To test this hypothesis, we used the pore-forming antibiotic
nystatin to introduce 4M
G into the cytosol of CHO-S cells under
conditions in which the cytosol remained relatively intact. Nystatin
forms pores in the plasma membrane which are permeable to univalent
ions and small neutral molecules (up to 8 Å diameter) but are
impermeable to proteins, multivalent ions, and organic molecules larger
than glucose (26, 27). CHO-S cells were pelleted and resuspended in KG
buffer that contained nystatin (0.24 mg/ml) followed by the addition of
puromycin and 4M
G. Both the puromycin-dependent and the
basal activation of 4M
G were observed in nystatin-perforated cells
(Fig. 7B). Compared with the values of
S0,basal and
S0,pur
measured with cells broken by N2 cavitation, the values of
S0,basal and
S0,pur
measured in nystatin-perforated cells were reduced by about 60-80%,
but their relative sizes were unchanged. The activation of 4M
G under these conditions was not the result of its entry into broken or otherwise damaged cells because 1 mM spermine did not
inhibit either
S0,pur or
S0,basal in nystatin-perforated cells (Fig. 7B) as it did in cells broken by cavitation. Spermine cannot
permeate nystatin pores, and the selective exclusion of spermine, but
not 4M
G, from nystatin-perforated cells was evidence that 4M
G was not activated by cells in which the plasma membrane had been
nonspecifically damaged. The preservation of similar characteristics in
the activation of 4M
G in cells perforated by nystatin compared with
cells broken by cavitation leads us to conclude that the activation of
4M
G in broken cells is unlikely to be an artifact resulting from the loss of critical cytosolic components.
 |
DISCUSSION |
We have demonstrated that a small polar dye, 4M
G, can serve as
a useful probe for studying the permeability of the ER membrane. In
gently broken CHO-S cells, the dye is activated by
-glucosidase II
located in the lumen of the ER. We report three key results in this
paper. First, treatment with puromycin significantly increased the rate
of activation of 4M
G in broken cells. Puromycin is highly selective
in terminating the elongation of proteins undergoing translation by
releasing nascent polypeptides from ribosomes (20, 21). In CHO-S cells,
this appeared to convert the pore of a ribosome-bound translocon from a
blocked state occupied by a nascent polypeptide to an empty state
permeable to 4M
G. This increased the number of empty translocon
pores available for the entry of dye because the majority of ribosomes
remain bound after the termination of translation by puromycin (25).
The specificity of puromycin's action provides strong evidence that
the rough ER is the site of activation of 4M
G because there is no
other site at which the release of nascent proteins from ribosomes
would increase the entry of 4M
G into a membrane-bound compartment.
For these reasons, we reject the hypothesis that translocon pores in
the ER membrane are impermeable to small, neutral molecules. Second, when puromycin-treated membranes were also treated with high salt, the
puromycin-dependent entry of 4M
G was abolished. Many
studies have demonstrated that a combined treatment with puromycin and high salt can dissociate ribosomes from ER membranes (23), and we
conclude that the loss of activation of 4M
G resulted from dissociation of the ribosomes from the translocons and closure of the
pores. The opposing effects of puromycin and high salt on the entry of
4M
G were very similar to the inhibition by high salt of the
puromycin-stimulated increase in the ionic permeability of translocons
incorporated into planar lipid bilayers (22).
A third key result was that there was a substantial basal rate of
activation of 4M
G in the absence of puromycin. We surmise from the
following evidence that permeation of translationally inactive,
ribosome-bound translocon pores accounts for most of the basal entry of
4M
G. The apparent Km values of the basal and
puromycin-dependent activation of 4M
G were increased by
an identical factor relative to the Km of the free
-glucosidase in detergent-permeabilized cells. This can be most simply accounted for by the puromycin-dependent and basal
entry of 4M
G into a membrane-bound compartment via pathways with the same permeability to 4M
G. The remarkable similarity in the
sensitivity of the puromycin-dependent entry and
approximately two-thirds of the basal entry to high salt provided
additional evidence that they shared a common, salt-sensitive pathway
for entry into the compartment. The puromycin-dependent
entry and the salt-sensitive component of the basal entry were both
partially inhibited by spermine, a putative pore-blocking compound.
Finally, the basal and puromycin-dependent entry of 4M
G
could be accounted for in a quantitative model based on the entry of
dye via empty, translationally inactive ribosome-translocon complexes.
Ribosomes remain bound to translocon pores for a sufficient period of
time after the termination of translation and the release of their
nascent polypeptide chains to provide a puromycin-independent pathway
for the entry of 4M
G. This interpretation is supported by a previous
study by Adelman et al. (23), who described two pools of
ribosomes bound to microsomes, a pool of translationally inactive
ribosomes or ribosomes with very short nascent chains that could be
stripped from microsomes by high salt treatment alone, and a pool of
translationally active ribosomes that could be released only by a
combined treatment with puromycin and high salt. Our conclusion is also
supported by recent reports demonstrating the persistent binding of the large subunit of ribosomes to the ER after the termination of translation (24, 25), and a model has been proposed in which the large
ribosomal subunit remains tightly bound to the translocon after the
termination of translation (7).
The translocon pore in CHO-S cells appears to have a higher
permeability to 4M
G than would be expected based on a model for the
regulation of ER permeability proposed by Johnson and colleagues (1, 2,
8, 28). They reported (2) that BiP maintains a tight permeability
barrier at the luminal end of the translocon pore in both its
ribosome-bound and unbound states. The barrier produced by BiP is
relaxed only when nascent peptides reach a sufficient length to extend
beyond the luminal end of the pore. We believe that our results can be
reconciled with Johnson's model by considering the following points.
First, Johnson's laboratory used only charged molecules to probe the
permeability of the ER, whereas we have used a polar, neutral molecule,
and it is possible that the permeability barrier presented by BiP might
be much smaller for uncharged molecules. What we describe as an
"open" pore permeable to 4M
G might not be permeable to ions. In
fact, inhibition of the entry of 4M
G by spermine is evidence that,
in our assay, the translocon pore is not permeable to a small,
polycationic molecule. This raises the possibility that the binding of
BiP might filter charged compounds, perhaps as a result of the
positioning of charged residues near the luminal mouth of the
translocon pore. Second, the conditions used in Johnson's assay would
have prevented the occurrence of translationally inactive, unoccupied
ribosomes bound to translocons. They used high salt/EDTA-stripped
microsomes from which all of the translationally inactive bound
ribosomes with empty pores would have been removed, and our experiments demonstrated that high salt significantly reduced the basal
permeability of the ER to 4M
G. The majority of bound ribosomes in
the assay by Johnson and co-workers were translationally active
ribosomes retaining a bound nascent polypeptide that could not be
released because of the absence of a stop codon in the mRNA used in
the in vitro translation reaction, and this would have
prevented any possible generation of empty translocons by the normal
release of the nascent polypeptide following termination of
translation. We did use a much simpler buffer than the wheat germ
lysate used by Johnson's laboratory, but the high permeability to
4M
G was not an artifact resulting from our use of this simple,
defined buffer, because a similar pattern of
puromycin-dependent and basal entry was observed in
nystatin-perforated cells, which would have retained most cytosolic
components. There is a precedent for our observation that the ER
membrane of CHO-S cells is permeable to 4M
G in an earlier report
that rough microsomes prepared from rat liver have a relatively high
permeability to a variety of small polar solutes, such as glucose
(29).
Our demonstration that empty translocons are permeable to 4M
G has
several broader implications. The evidence that the permeability of a
translocon pore to a neutral solute is different from its permeability
to ions suggests that further study should be directed at how permeant
molecules are filtered, or selected, as they pass through the pore of
the translocon. Also, recent studies have revealed that the translocon
pore mediates the post-translational export, as well as cotranslational
import, of many proteins (6). An important implication of this broader
role of the translocon in protein transport is that the pore is a very
busy thoroughfare, which again raises the question of how the
permeability of the translocon pore to a broad spectrum of molecules is
regulated. Further study of the entry of 4M
G should be useful in
defining the mechanisms that regulate the passage of small, uncharged
molecules across the ER membrane under various conditions. Finally, the presence of a basal permeability to 4M
G in the absence of puromycin is especially intriguing because permeation of the translocon pore by
small, neutral molecules might convey important signals between the
lumen of the ER and the cytosol.
 |
ACKNOWLEDGEMENTS |
We thank Drs. James Mahaney and Drew
Shiemke for comments on a draft of this manuscript.
 |
FOOTNOTES |
*
This work was supported by American Heart Association Grant
AHA 0051268B (to W. F. W.).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, April 12, 2001, DOI 10.1074/jbc.M102409200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
BiP, immunoglobulin-binding protein;
4M
G, 4-methylumbelliferyl
-D-glucopyranoside;
4M
G, 4-methylumbelliferyl
-D-glucopyranoside;
CHO-S cells, Chinese hamster ovary-S cells;
S0, initial
slope;
S0, basal, puromycin-independent slope;
S0, pur, slope in the presence of puromycin;
S0, pur, increase of
S0,pur above S0,basal;
Stotal, maximum number of empty pores in the
presence of puromycin;
Sres, background
activation.
 |
APPENDIX |
The starting point for the calculation is an estimate of the total
number of translocon pores that can be found in the empty state. The
analysis is limited to the dynamic pool of pores that can be in the
empty state either before or after treatment with puromycin. We assume
that puromycin can convert all translationally active pores, blocked by
nascent polypeptides, into empty pores. Therefore, in the presence of
puromycin, the rate of activation of 4M
G caused by entry through the
maximum number of empty pores can be estimated by dividing the
salt-sensitive fraction of S0,pur by the
fraction, s, of ribosome-bound pores that can be closed by
high salt
|
(A1)
|
All other components are calculated from
Stotal.
In the Presence of Puromycin--
In normal salt,
|
(A2)
|
where Sres is a salt-insensitive background
activation of 4M
G of unknown origin, assumed to be 2% of the
detergent control (see Fig. 7B).
In high salt,
|
(A3)
|
where (1
s) is the salt-insensitive fraction.
In the Absence of Puromycin--
In normal salt,
|
(A4)
|
where f is the fraction of the pores that are
empty in the absence of puromycin, and x is the permeability
of a blocked pore to 4M
G as a fraction of the permeability of an
empty pore.
In high salt,
|
(A5)
|
 |
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