Am J Physiol Cell Physiol 291: C511–C517, 2006.
First published April 12, 2006; doi:10.1152/ajpcell.00274.2005.
Translocon pores in the endoplasmic reticulum are permeable to small anions
Beáta Lizák,1 Ibolya Czegle,1 Miklós Csala,1,2 Angelo Benedetti,3 József Mandl,1,2 and Gábor Bánhegyi1,2,3
1
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 2Endoplasmic
Reticulum Research Group of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary;
and 3Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanità Pubblica, Università di Siena, Siena, Italy
Submitted 10 June 2005; accepted in final form 31 March 2006
puromycin; UDP-glucuronosyltransferase; glucose-6-phosphatase;
␤-glucuronidase
of the endoplasmic reticulum
(ER) gain their substrate supply from the cytosol. The substrates are often anions, which cannot cross the membrane by
simple diffusion. Specific transporters may facilitate their
movement across the membrane, although only a few ER
transporters have been characterized at the molecular level (6,
20, 22). It has been repeatedly observed that microsomal
vesicles derived from the ER exhibit a basal permeability
toward various compounds, including xenogenous molecules,
which presumably do not have strictly specific transporters.
This nonspecific permeability is usually attributed to the damage or to the improper orientation of the membrane. However,
recent observations indicate that translocon protein channels
may play a role in the permeation of small molecules across the
ER membrane.
SEVERAL INTRALUMINAL ENZYMES
Address for reprint requests and other correspondence: G. Bánhegyi, Dept.
of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis Univ., PO Box 260, 1444 Budapest, Hungary (e-mail: banhegyi@puskin.
sote.hu).
http://www.ajpcell.org
Co-translational protein translocation and integration into the
membrane of the ER occur at sites termed translocons (24, 28,
35–37). The translocon is composed of several ER membrane
proteins that are associated to form an aqueous pore. This pore
must maintain a permeability barrier because calcium ion is stored
in the ER and the unregulated release of this potent second
messenger would abolish a fundamental signaling mechanism of
the cell. During co-translational translocation, the aqueous translocon pore is sealed at its cytoplasmic end by the growing
polypeptide chain (35). The permeability of the translocon has
been proposed to be regulated at its luminal side by the prominent
ER chaperone BiP, a protein released from the translocon shortly
after the completion of ribosome nascent chain targeting. In its
empty state, when the translocon pore is ribosome-bound but
unoccupied by polypeptide, the complex seems to allow the
passage of small molecules (18, 33, 35).
Lomax et al. (27) studied the Ca2⫹ leak pathways in the ER
of pancreatic acinar cells by directly measuring [Ca2⫹] in the
ER. It was demonstrated that the leak was not blocked by either
the IP3 receptor antagonist heparin or the ryanodine receptor
antagonist ruthenium red, but they found a puromycin-induced
Ca2⫹ efflux from the ER. Puromycin is an antibiotic that
selectively terminates ribosomal translation by releasing the
nascent polypeptide from the protein channel of the ribosome
(32, 35, 38).
Wonderline and coworkers (18, 33) tested the permeation of
a small neutral molecule, 4-methyl-umbelliferyl-␣-D-glucopyranoside, by measuring its hydrolysis by an ER resident ␣-glucosidase, which is dependent on its entry into the ER. They
found that translocons are permeable to this neutral molecule,
as long as empty ribosomes remain bound to them.
It has been observed that low molecular weight anions,
which presumably do not have specific ER transporters, e.g.,
mannose-6-phosphate, can cross the ER membrane, although
with a moderate speed. Therefore, the aim of the present study
was to investigate the permeation of small anions through translocon channels in rat liver microsomal vesicles. The transport was
studied by two methods: 1) by comparing the activity of enzymes
with intraluminal active sites in intact and permeabilized microsomes and 2) by directly examining the transport of substrates
using the light scattering and rapid filtration techniques. The
results confirmed that translocon channels can contribute to the
permeation of small anions across the ER membrane.
MATERIALS AND METHODS
Preparation of rat liver microsomes. Liver microsomes were prepared from fed male Sprague-Dawley rats (180 –230 g body wt;
The costs of publication of this article were defrayed in part by the payment
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0363-6143/06 $8.00 Copyright © 2006 the American Physiological Society
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Lizák, Beáta, Ibolya Czegle, Miklós Csala, Angelo Benedetti,
József Mandl, and Gábor Bánhegyi. Translocon pores in the endoplasmic reticulum are permeable to small anions. Am J Physiol Cell
Physiol 291: C511–C517, 2006. First published April 12, 2006;
doi:10.1152/ajpcell.00274.2005.—Contribution of translocon peptide
channels to the permeation of low molecular mass anions was investigated in rat liver microsomes. Puromycin, which purges translocon
pores of nascent polypeptides, creating additional empty pores, raised
the microsomal uptake of radiolabeled UDP-glucuronic acid, while it
did not increase the uptake of glucose-6-phosphate or glutathione. The
role of translocon pores in the transport of small anions was also
investigated by measuring the effect of puromycin on the activity of
microsomal enzymes with intraluminal active sites. The mannose-6phosphatase activity of glucose-6-phosphatase and the activity of
UDP-glucuronosyltransferase were elevated upon addition of puromycin, but glucose-6-phosphatase and ␤-glucuronidase activities were
not changed. The increase in enzyme activities was due to a better
access of the substrates to the luminal compartment rather than to
activation of the enzymes. Antibody against Sec61 translocon component decreased the activity of UDP-glucuronosyltransferase and
antagonized the effect of puromycin. Similarly, the addition of the
puromycin antagonist anisomycin or treatments of microsomes, resulting in the release of attached ribosomes, prevented the puromycindependent increase in the activity. Mannose-6-phosphatase and UDPglucuronosyltransferase activities of smooth microsomal vesicles
showed higher basal latencies that were not affected by puromycin. In
conclusion, translationally inactive, ribosome-bound translocons allow small anions to cross the endoplasmic reticulum membrane. This
pathway can contribute to the nonspecific substrate supply of enzymes
with intraluminal active centers.
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radioactivity (3). The alamethicin-permeabilized microsomes were
recovered on filters and washed as above. More than 95% of microsomal protein was retained by filters in cases of both native and
alamethicin-permeabilized vesicles, indicating that the alamethicin
treatment did not affect the vesicular structure of microsomes. The
alamethicin-permeabilized microsomes retained amounts of radioactivity ⬍20% of that associated with untreated microsomes. Intravesicular radioactive compounds were bona fide lost during the washing
procedure because the alamethicin nonreleasable portion did not
further decrease even after extensive washing of filters (and microsomes). This allowed us to regard the alamethicin-releasable portion
of radioactivity as intravesicular.
Enzyme activities. All enzymatic assays were performed in buffer A
containing 1 mg/ml microsomal protein at 37°C. Permeabilized microsomal vesicles were treated with the pore-forming alamethicin
(0.05 mg/mg protein) immediately before the assay. UDP-glucuronosyltransferase activity was measured by using p-nitrophenol as substrate (0.5 mM) in the presence of 3 mM UDP-glucuronic acid.
Absorbance of p-nitrophenol was measured spectrophotometrically at
400 nm, at time 0 and 10 min of incubations. Activity was calculated
on the basis of p-nitrophenol disappearance. Glucose-6-phosphatase
activity was measured in the presence of 1 mM glucose-6-phosphate
or mannose-6-phosphate for 30 min. The phosphate released by the
enzymes was measured with the malachite green reagent (31). Absorbance at 660 nm was measured and compared with sodium phosphate standards. ␤-Glucuronidase activity was assayed by using either
p-nitrophenyl glucuronide or phenolphthalein glucuronide (1–1 mM)
as substrate (10). Incubation time was 30 min; the corresponding
deliberated aglycones were measured after alkalinization of samples
at 400 and 540 nm, respectively.
Immunoblot analysis. Microsomal proteins were separated by 11%
SDS-PAGE and transferred to PVDF filter membranes by electroblotting. Lowfat milk (5%) in PBS containing 0.5% (vol/vol) Tween
20 solution was used for blocking and for dissolving the primary
antibody, which was applied for 1 h at room temperature. Anti-Sec61
(catalog no. PA3-014, Affinity Bioreagents) rabbit polyclonal antibody was detected using a fluorescein-conjugated anti-rabbit Igspecific secondary antibody. To amplify the signal an anti-fluorescein
alkaline phosphatase-conjugated tertiary antibody was also used (all
antibodies were purchased from Amersham Biosciences). Blots were
overlaid with the fluorescent ECF substrate (Amersham Biosciences).
Fluorescence was detected using a fluorescence imaging system
(Typhoon, Molecular Dynamics). The primary antibody labeled a
band of the expected molecular weight (38 kDa).
Statistical analyses. Statistical comparisons were made by
ANOVA, followed by a Dunnett’s test (Table 1) or by a Tukey’s test
(Table 3). In the case of Tables 2 and 4 and Fig. 1, paired two-tailed
Student’s t-test was used.
Chemicals. Glucose-6-phosphate (monosodium salt), mannose-6phosphate (disodium salt), glutathione, UDP-glucuronic acid, p-nitrophenyl glucuronide, phenolphthalein glucuronide, alamethicin, puromycin, anisomycin, and 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic
acid were obtained from Sigma. D-[14C]Glucose-6-phosphate (300
mCi/mmol) and [14C]sucrose (500 mCi/mmol) were from American
Radiolabeled Chemicals (St. Louis, MO). [14C]UDP-glucuronic acid
(2.2 mCi/mmol) was purchased from UVVVR (Prague, Czech Republic). [3H]Glutathione was obtained from NEN Life Science Products (Boston, MA). Dithiothreitol was removed from the [3H]glutathione solution by extraction as described by Butler et al. (9). Cellulose acetate/nitrate filter membranes (pore size 0.22 ␮m) were from
Millipore. All other chemicals were of analytical grade.
RESULTS
Puromycin increases permeability of rat liver microsomes to
citrate and sucrose. In the first set of experiments, the effect of
puromycin was validated by using membrane nonpermeant
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Charles River Hungary) as reported earlier (3). The livers were
homogenized in sucrose-HEPES buffer (0.3 M sucrose and 0.02 M
HEPES, pH 7.2) with a glass-Teflon homogenizer. The microsomal
fraction was then isolated using fractional centrifugation. After centrifugation of liver homogenates for 10 min at 1,000 g, the supernatant
was spun for 10 min at 12,000 g. The 12,000 g supernatant was
centrifuged for 60 min at 100,000 g; the sediment was resuspended in
buffer A containing (in mM) 100 KCl, 20 NaCl, 1 MgCl2, and 20
4-morpholinepropanesulfonic acid, pH 7.2, and was centrifuged again for
60 min at 100,000 g. The final pellet (microsomes) was resuspended in
buffer A to give ⬃50 mg/ml protein concentration, then immediately
frozen in liquid nitrogen and kept in liquid nitrogen until use (within 2
mo). The microsomal fraction was characterized by measuring marker
enzyme activities (5). The activity of cytochrome c oxidase, glucose-6phosphatase, and 5⬘-nucleotidase in the microsomal fraction was 1.2 ⫾
0.3, 30.1 ⫾ 5.0, and 6.0 ⫾ 1.1, respectively, expressed as percents of
activities measured in total homogenate (means ⫾ SD, n ⫽ 3).
Rough and smooth subfractions of liver microsomes were prepared
according to (5). The purity of the subfractions was evaluated by
immunoblot analysis of Sec61␣. Compared with the total microsomal
fraction, 1.5-fold enrichment in the rough and fivefold impoverishment in the smooth subfraction was observed by the densitometry of
the corresponding bands.
Integrity of microsomal vesicles was assessed by measuring the
latency of mannose-6-phosphatase activity (8). Protein concentration
of microsomes was determined using Bio-Rad protein assay with
bovine serum albumin as a standard, according to the manufacturer’s
instructions.
Transport measurements by light-scattering technique. The permeability of the microsomal membranes to sucrose was also measured by
continuous detection of the osmotically induced changes in size and
shape of microsomal vesicles (3, 25, 29). Briefly, ER vesicles (50
␮g/ml protein) were equilibrated for 2 h in a hypotonic medium (5
mM 1,4-piperazine diethanesulfonic acid potassium salt, pH 7.0).
Light scattering of the microsomal suspensions was then monitored at
400 nm with the use of a fluorimeter (Hitachi F-4500) equipped with
a temperature-controlled cuvette holder (22°C) and magnetic stirrer.
The addition of a small volume (⬍5% of the total incubation volume)
of a concentrated osmolyte solution results in a rapid increase in light
scattering (due to the shrinkage of the vesicles). This peak is followed
by a gradual decrease in light scattering (which reflects vesicle
swelling due to progressive equilibration of the osmolyte concentration between the extra- and intravesicular spaces). The relative height
of the peak and the slope of the curve depend on the permeability of
the vesicular membrane. Light-scattering registrations were terminated by the addition of the pore-forming compound alamethicin. The
channels formed by alamethicin have been estimated to be 10 Å in
diameter with no real selectivity between univalent ions. Alamethicin
channels allow the passage of low-molecular-mass compounds such
as dicarboxylates and nicotinamide nucleotides, but exclude folded
proteins (Ref. 23 and references therein). Upon alamethicin addition,
the signal returned to the basal level, indicating that the osmotic
effects did not alter the vesicular structure of the microsomes.
Rapid filtration experiments. For the measurement of the uptake
and the accumulation of radiolabeled compounds, liver microsomes (1
mg protein/ml) were incubated in buffer A containing 0.02 mM
UDP-glucuronic acid, sucrose, glucose-6-phosphate, or 0.4 mM glutathione plus their radiolabeled analogues (1 ␮Ci/ml [14C]UDPglucuronic acid, 1 ␮Ci/ml [14C]sucrose, 10 ␮Ci/ml [3H]glutathione, 2
␮Ci/ml D-[14C]glucose-6-phosphate) at 22°C. At the indicated time
points, samples (0.1 ml) were rapidly filtered through cellulose acetate/nitrate filter membranes (pore size: 0.22 ␮m) and were washed
with ice-cold buffer A containing 1 mM 4,4⬘-diisothiocyanostilbene2,2⬘-disulfonic acid. The radioactivity associated with microsomes
retained by filters was measured by liquid scintillation counting. In
each experiment, alamethicin (0.05 mg/mg protein) was added to
parallel incubates to distinguish the intravesicular and the bound
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Table 1. Effect of puromycin on microsomal uptake of
sucrose, glucose-6-phosphate, and glutathione
Addition
n
Uptake,
pmol/mg
protein
Sucrose (0.02 mM)
Sucrose ⫹ puromycin
Sucrose (smooth microsomes)
Sucrose ⫹ puromycin (smooth microsomes)
Glucose-6-phosphate (0.02 mM)
Glucose-6-phosphate ⫹ puromycin
Glutathione (0.4 mM)
Glutathione ⫹ puromycin
6
6
6
6
4
4
4
4
23.7⫾1.4
28.4⫾2.7*
11.8⫾0.8*
11.3⫾3.5
39.9⫾5.2
42.8⫾1.5
32.6⫾1.6
36.9⫾2.4
compounds. Sucrose and citrate are not metabolized inside the
ER; therefore, presumably they do not have specific membrane
transporters. The permeation of these compounds was checked
by the light scattering method. Addition of sucrose or citrate to
microsomal suspensions resulted in a rapid increase in the light
scattering signal reflecting a shrinkage of vesicles, which was
followed by a very slow decrease in the signal reflecting a the
swelling phase due to the influx (Fig. 1). The slope of the
second phase was steeper in microsomes preincubated with
puromycin (0.5 mM), indicating that puromycin increased the
permeability of the vesicles towards both sucrose and citrate
(Fig. 1). The calculated slope values upon addition of sucrose
were ⫺2.26 ⫾ 0.17 (control) and ⫺4.23 ⫾ 0.30 (puromycin)
upon addition of citrate were ⫺0.41 ⫾ 0.28 (control) and
⫺0.78 ⫾ 0.19 (puromycin). Values are expressed as arbitrary
units per minute, means ⫾ SD. In both cases, the slope in
puromycin-treated samples differed significantly from that of
controls (P ⬍ 0.01). Puromycin concentrations ⬎0.5 mM did
not result in a more pronounced effect (data not shown).
Fig. 1. Effect of puromycin on sucrose and citrate uptake in rat liver microsomes.
Microsomal vesicles (50 ␮g protein/ml) were equilibrated for 2 h in a hypotonic
medium. Then the light scattering of the microsomes was monitored at 22°C. At the
indicated time (solid arrow) a small volume (2.5% of the total incubation volume) of
a concentrated solution of sucrose (top) or citrate (bottom) was added to reach 50 mM
final concentration. Puromycin (0.5 mM) was added immediately before the osmolytes. Trace a, control; trace b, puromycin addition. At the end of the registrations
the pore-forming compound alamethicin (A, open arrow; 0.05 mg/mg protein) was
added. Representative traces of 4–9 independent registrations are shown.
To control whether this increase in permeability can really
be attributed to the translocon pore (and not to a nonspecific
effect on the membrane), the effect of puromycin was evaluated either in total (nonfractionated) and smooth microsomes,
Table 2. Effect of puromycin on activity of intraluminal ER enzymes
Native Microsome Activity,
nmol䡠min⫺1䡠mg protein⫺1
Enzyme
G6Pase
UGT
␤-Glucuronidase
Permeabilized Microsome
Activity, nmol䡠min⫺1䡠mg
protein⫺1
⫺P
⫹P
Increase in
Activity,
nmol䡠min⫺1䡠mg
protein⫺1
79.0⫾3.6
81.1⫾8.8
90.9
73.4
89.4
75.6
1.2
0
32.5⫾1.1
30.6⫾0.3
90.7
87.4
0.86
0.86⫾0.08
1.33⫾0.18
0.84⫾0.04
1.20⫾0.02
20.9
21.8
28.6
16.7
0
0
Substrate, mM
⫺P
⫹P
⫺P
M6P (1)
G6P (1)
pNP (0.5)
UDPGA (3)
pNPG (1)
phenG (1)
7.19⫾0.06
20.1⫾1.7
8.39⫾0.23†
19.8⫾1.4
79.3⫾3.6
75.7⫾2.9
3.01⫾0.19
3.87⫾0.48*
0.68⫾0.06
1.04⫾0.04
0.60⫾0.04
1.00⫾0.04
⫹P
%Latency
Values are means ⫾ SD; n ⫽ 6 samples. P, puromycin; UGT, UDP-glucuronosyltransferase; ER, endoplasmic reticulum; M6P, mannose-6-phosphate; G6P,
glucose-6-phosphate; pNP, p-nitrophenol; UDPGA, UDP-glucuronic acid; pNPG, p-nitrophenyl glucuronide; phenG, phenolphthalein glucuronide. Rat liver
microsomal vesicles (1 mg protein/ml) were incubated in buffer A in the presence or in absence of 0.5 mM puromycin at 37°C. Enzymatic activities were
measured at the indicated substrate concentrations, as described in MATERIALS AND METHODS. Permeabilized vesicles were treated with alamethicin (0.05 mg/mg
protein). *P ⬍ 0.05; †P ⬍ 0.01, statistically significant differences vs. untreated control.
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Values are means ⫾ SD; n, no. of samples. Values measured in alamethicintreated samples were regarded as binding and were subtracted. Nonfractionated
microsomal vesicles and smooth microsomes (where indicated) (1⫺1 mg
protein/ml) were incubated in buffer A in the presence of sucrose, glucose-6phosphate, or glutathione plus radioactive tracer ([14C] sucrose and glucose6-phosphate, [3H]glutathione) at 22°C. Puromycin (0.5 mM) was added immediately before sucrose. Parallel samples were treated with the pore-forming
compound alamethicin (0.05 mg/mg protein). After 5 min of incubation,
0.1-ml samples were taken and filtered, as described in MATERIALS AND
METHODS. The radioactivity associated with microsomes was measured by
liquid scintillation counting. *P ⬍ 0.01, statistically significant difference vs.
sucrose.
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Fig. 2. Effect of puromycin on microsomal UDP-glucuronic acid uptake.
Microsomal vesicles (1 mg protein/ml) were incubated in buffer A in the
absence (■) or in the presence (Œ) of 0.5 mM puromycin at 22°C. The
incubations also contained 0.02 mM UDP-glucuronic acid plus radioactive
tracer ([14C]UDP-glucuronic acid) At the indicated times, samples (0.1 ml)
were taken and filtered as described in MATERIALS AND METHODS. The radioactivity associated with microsomes was measured by liquid scintillation
counting. Time 0 values were regarded as binding and were subtracted. Data
are means ⫾ SD, n ⫽ 6.
AJP-Cell Physiol • VOL
Fig. 3. Effect of puromycin on microsomal UDP-glucuronosyltransferase,
mannose-6-phosphatase, and glucose-6-phosphatase activities. Microsomal
vesicles (1 mg microsomal protein/ml) were incubated in buffer A at 37°C.
UDP-Glucuronosyltransferase activity (■) was measured by using p-nitrophenol as substrate (0.5 mM) in the presence of 3 mM UDP-glucuronic acid.
Absorbance of p-nitrophenol was measured, and the activity was calculated on
the basis of p-nitrophenol disappearance. Mannose-6-phosphatase (Œ) and
glucose-6-phosphatase (F) activities were detected in the presence of 1 mM
substrate on the basis of phosphate measurement. Data are expressed as a
percentage of control, means ⫾ SD, n ⫽ 6. Control values: UDP-glucuronosyltransferase activity 3.01 ⫾ 0.19, mannose-6-phosphatase activity 7.19 ⫾
0.06 glucose-6-phosphatase activity 20.1 ⫾ 1.7 nmol 䡠 min⫺1 䡠 mg protein⫺1.
vesicles. Agents that disrupt the integrity or increase the
permeability of the membrane cause an increase in the activity
of these enzymes.
Microsomal UDP-glucuronosyltransferase activity was measured by using p-nitrophenol and UDP-glucuronic acid as
substrates. The activity in native microsomal vesicles was less
than 10% of the total activity measured in permeabilized
vesicles (Table 2). The addition of puromycin resulted in a
concentration-dependent increase in the activity (Fig. 3); 30%
activation was observed at 0.5 mM puromycin concentration.
Puromycin concentrations ⬎0.5 mM did not cause a more
pronounced effect (data not shown). However, puromycin was
ineffective in alamethicin-permeabilized microsomes (Table
2), which indicates that the effect was likely due to the
improved access of UDP-glucuronic acid the enzyme to the
enzyme and not a direct activation of the enzyme itself.
Microsomal ␤-glucuronidase activity was measured with
p-nitrophenyl glucuronide and phenolphthalein glucuronide as
substrates. In accordance with previous results (10), the enzyme had a moderate latency in control vesicles. Puromycin
did not increase ␤-glucuronidase activity (Table 2).
The catalytic subunit of glucose-6-phosphatase is able to
hydrolyze hexose phosphates other than glucose-6-phosphate
(41). The specificity of glucose-6-phosphatase activity is guaranteed by an ER transport protein, which is strictly specific to
glucose-6-phosphate (14, 15, 19). Consequently, the latency of
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in which translocons are underrepresented. The uptake was
measured with rapid filtration technique, by using radiolabeled
sucrose. The basal rate of sucrose uptake was higher in total
compared with smooth microsomes, calculated either on protein (Table 1) or on phospholipid basis (data not shown).
Puromycin (0.5 mM) caused an increase in the rate of sucrose
uptake of total microsomes only (Table 1).
Puromycin increases uptake of UDP-glucuronic acid in rat
liver microsomes. The uptake of various anions by rat liver
microsomes was detected by using radiolabeled compounds by
rapid filtration. The control time course of UDP-glucuronate
(0.02 mM) influx showed the characteristic initial overshoot,
followed by a decline, as it has been reported by others (7). A
completely different curve was gained in the presence of
puromycin (0.5 mM); both the overshoot and the declination
phases disappeared and an equilibrating uptake was detected
(Fig. 2). Puromycin was ineffective in case of glucose-6phosphate and glutathione transport (Table 1). In case of
glutathione a higher (0.4 mM) concentration should had to be
used; at comparable (0.02 mM) concentration uptake could not
be properly detected due to a high background caused by
binding.
Puromycin promotes substrate access to intraluminal enzymes of ER. UDP-glucuronosyltransferases (13), glucose-6phosphatase (41), and ␤-glucuronidase (42) are ER enzymes
with active sites located in the lumen. Hence, the ER membrane represents a barrier for their substrates; consequently, the
activity of these enzymes is reduced in native microsomal
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Table 3. Prevention of effect of puromycin
UDP-Glucuronosyltransferase
Activity, %Control
Treatment
100.0⫾19.6
129.8⫾18.8*
50.0⫾22.0*
81.0⫾27.0†
107.0⫾16.0‡
118.6⫾21.7
94.3⫾13.6‡
139.6⫾9.3§
63.2⫾25.9*
55.3⫾18.7
98.8⫾19.1
107.3⫾17.8
98.9⫾21.2
101.9⫾22.3†
132.9⫾31.7
Data are means ⫾ SD; n ⫽ 6 –32. Rat liver microsomal vesicles (1 mg
protein/ml) were incubated in buffer A at 22°C. The concentrations of various
agents were 0.1 mM puromycin, 0.05 mM anisomycin, and 5 mM EDTA.
High-salt buffer contained 20 mM KCI in buffer A. In case of pretreatments,
microsomes were preincubated for 10 min with native or denatured anti-Sec61
antibody, anti-rabbit secondary antibody, anisomycin, or puromycin. AntiSec61 antibody was denatured by boiling for 5 min. UDP-glucuronosyltransferase activities were measured by using p-nitrophenol as substrate (0.5 mM)
in the presence of 3 mM UDP-glucuronic acid. Absorbance of p-nitrophenol
was measured, and the activity was calculated on the basis of p-nitrophenol
disappearance. Control UDP-glucuronosyltransferase activity was 2.99 ⫾ 0.60
nmol䡠min⫺1 䡠 mg protein⫺1. Statistically significant differences were the following: *P ⬍ 0.001 vs. untreated control; †P ⬍ 0.001 vs. puromycin treated;
‡P ⬍ 0.05 vs. anti-Sec61 treated; §P ⬍ 0.05 vs. anti-rabbit secondary antibody
treated.
the enzyme is lower with glucose-6-phosphate as substrate than
with other hexose phosphates, e.g., with mannose-6-phosphate
(8). Therefore, changes in hexose-6-phosphatase activities upon
puromycin addition were monitored by using both substrates.
The addition of puromycin resulted in a concentrationdependent increase in mannose-6-phosphatase activity (Fig. 3).
A maximal activation of ⬃15% was observed. Puromycin was
ineffective in alamethicin-permeabilized microsomes (Table
2), which argues against a direct activation of the enzyme.
Puromycin was unable to increase the activity when glucose6-phosphate was used as substrate (Table 2 and Fig. 3).
Table 4. Comparison of enzyme activities and latencies in microsomal vesicles derived from rough or smooth ER
Native Microsome Activity,
nmol䡠min⫺1䡠mg protein⫺1
Enzyme Substrate mM
Glucose-6-phosphatase
M6P (1)
G6P (1)
UDP-glucuronosyltransferase
pNP (0.5)
UDPGA (3)
RER
SER
Permeabilized Microsome
Activity, nmol䡠min⫺1䡠mg
protein⫺1
RER
SER
%Latency
RER
SER
Difference in Latency
8.5⫾0.3
19.0
7.5⫾0.2†
20.1
68.8⫾1.4
81.3
70.9⫾2.3
86.7
87.7
76.6
90.1
76.8
2.4
0.2
3.20⫾0.28
3.63⫾0.37*
19.5⫾2.8
45.1⫾2.1†
83.6
91.9
8.3
Data are means ⫾ SD; n ⫽ 6 (for G6P, n ⫽ 2). Rough (RER) or smooth (SER) fractions of rat liver microsomal vesicles (1 mg protein/ml) were incubated
in buffer A at 37°C. Enzymatic activities were measured at the indicated substrate concentrations, as described in MATERIALS AND METHODS. Permeabilized vesicles
were treated with alamethicin (0.05 mg/mg protein). *P ⬍ 0.05; †P ⬍ 0.01, statistically significant differences vs. RER.
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Effect of anti-Sec61 antibody
None
Puromycin
Anti-Sec61
Anti-Sec61 then puromycin
Denatured anti-Sec61
Denatured anti-Sec61 then puromycin
Anti-rabbit secondary antibody
Anti-rabbit secondary antibody then
puromycin
Effect of ribosome release
EDTA
EDTA then puromycin
High-salt buffer
High-salt buffer then puromycin
Effect of anisomycin
Anisomycin
Anisomycin then puromycin
Puromycin then anisomycin
Effect of puromycin on UDP-glucuronosyltransferase activity can be prevented by a translocon-binding antibody, by
puromycin antagonist anisomycin, and by release of ribosomes. It has been reported that an antibody (or its proteolytic
fragment) binding to the Sec61 protein of the translocon
occludes the pore of the channel (17). Therefore, the translocon-specific effect of puromycin was controlled by investigating the effect of this antibody on UDP-glucuronosyltransferase
activity. Specific binding of the antibody was checked by
immunoblot analysis of the microsomal proteins (data not
shown). As expected, the addition of the antibody to microsomes counteracted the effect of puromycin. Moreover, it
decreased the activity of UDP-glucuronosyltransferase even in
the absence of puromycin (Table 3). The inhibition was specific, since neither the heat-denatured antibody, nor an antirabbit secondary antibody, was effective (Table 3).
Anisomycin is a known antagonist of puromycin (21), which
is effective only if added beforehand. Anisomycin has been
successfully used in microsomal experiments to prevent various translocon-dependent effects of puromycin (27, 33). In our
experiments, puromycin failed to increase UDP-glucuronosyltransferase activity when added after anisomycin. If anisomycin was added after puromycin, it was ineffective (Table 3).
The release of ribosomes from the surface of microsomal
vesicles also prevented the effect of puromycin. Pretreatment
with EDTA (5 mM), which is known to detach ribosomes (12),
decreased UDP-glucuronosyltransferase activity by 37%, and
completely abolished the effect of puromycin. As shown in
Table 3, puromycin was also ineffective in microsomes pretreated with a high-salt buffer, another maneuver that also
removes ribosomes from the microsomal membrane (30).
Treatment of native microsomes with high-salt buffer did not
affect UDP-glucuronosyltransferase activity; in permeabilized
microsomes the buffer slightly increased the activity (data not
shown).
Different membrane permeability in smooth and rough microsomes. Because recent studies (30, 34), have indicated that
nontranslating empty ribosomes are present on the surface of
the ER, this condition may therefore contribute to the permeability of the ER membrane. By using the above approach, we
compared the latency of UDP-glucuronosyltransferase, glucose-, and mannose-6-phosphatase activities in rough (i.e.,
translocon rich) and smooth microsomal fractions. All of these
enzyme activities were present in both microsomal fractions
(Table 4). The latency of UDP-glucuronosyltransferase activity
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ANION TRANSPORT BY TRANSLOCON
was much greater in smooth vesicles. The latency of glucose6-phosphatase enzyme measured with glucose-6-phosphate
was similar in the two fractions. However, when mannose-6phosphate was added as substrate, a higher latency was found
in smooth microsomes (Table 4).
DISCUSSION
AJP-Cell Physiol • VOL
GRANTS
This work was supported by the Italian Ministry of University and Research
Grant RBAU014PJA, the Hungarian Ministry of Health Grants ETT 090/2003
and 613/2003, National Scientific Research Fund Grants OTKA F37484,
F46740, T48939, T38312, and TS49851, and a grant from University of Siena
(quota progetti; to A. Benedetti).
REFERENCES
1. Bánhegyi G, Braun L, Marcolongo P, Csala M, Fulceri R, Mandl J,
and Benedetti A. Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles. Biochem J 315: 171–176,
1996.
2. Bánhegyi G, Lusini L, Puskás F, Rossi R, Fulceri R, Braun L, Mile V,
di Simplicio P, Mandl J, and Benedetti A. Preferential transport of
glutathione versus glutathione disulfide in rat liver microsomal vesicles.
J Biol Chem 274: 12213–12216, 1999.
3. Bánhegyi G, Marcolongo P, Fulceri R, Hinds C, Burchell A, and
Benedetti A. Demonstration of a metabolically active glucose-6-phosphate pool in the lumen of liver microsomal vesicles. J Biol Chem 272:
13584 –13590, 1997.
4. Battaglia E and Gollan J. A unique multifunctional transporter translocates estradiol-17␤-glucuronide in rat liver microsomal vesicles. J Biol
Chem 276: 23492–23498, 2001.
5. Benedetti A, Fulceri R, Romani A, and Comporti M. MgATP-dependent glucose 6-phosphate-stimulated Ca2⫹ accumulation in liver microso-
291 • SEPTEMBER 2006 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on July 31, 2017
The present findings show that translocon protein channels
can mediate the permeation of small molecular weight anions
(besides calcium ions and neutral polar molecules) across the
ER membrane. As reported previously (35, 36), the translocon
may permit the transmembrane flux of small anions, such as
glutamate and HEPES. In the present study, the membrane
permeation of six anions of different size and shape (glucose6-phosphate, mannose-6-phosphate, glutathione, UDP-glucuronic acid, phenolphthalein glucuronide, and p-nitrophenyl
glucuronide) was examined. All of these compounds are (or
can be) metabolized by enzymes with an intraluminal activity,
therefore, a possible contribution of the translocon channel to
their membrane transport has a physiological relevance.
Puromycin was used to empty the translocon channel by
releasing the incomplete nascent polypeptide from the ribosome. The effect of puromycin was specific because 1) it was
absent in the smooth microsomal subfraction poor in translocons (Table 1.); 2) it did not cause a generalized permeability
of the vesicles (Fig. 3 and Table 2); 3) it was ineffective in
permeabilized vesicles (Table 2); and 4) it could be antagonized by anti-Sec61 antibody, by anisomycin, and by the
release of ribosomes from the membrane (Table 3).
The puromycin concentrations used in our study were similar to those in previous studies, and the effective concentrations were in good agreement with other observations. In a
recent study, Van Coppenolle et al. (40) applied 200 ␮M
puromycin. Others have used similar concentrations (100 –
1,000 ␮M) to estimate the puromycin effect on ER permeability (18, 27, 33).
Although the pore of the translocon complex in the ER is
large enough to be permeated by small molecules, it is generally believed that permeation is prevented by a barrier constituted by BiP at the luminal end of the pore (17). A recent study
(18) rejected this assumption and concluded that a small,
neutral molecule can permeate the empty pore of a translationally inactive, ribosome-bound translocon even in the presence
of luminal proteins. Our findings support this observation and
affirm that the translocon could provide a pathway for small
neutral and anionic molecules to cross (although slowly) the
ER membrane.
The effect of puromycin on anion transport is controversial
at first sight. The permeation of mannose-6-phosphate and
UDP-glucuronic acid was increased upon puromycin addition
(Figs. 2 and 3 and Table 2), whereas in case of glucose-6phosphate, glutathione, p-nitrophenyl glucuronide and phenolphthalein glucuronide, no effect could be demonstrated (Fig. 3,
Tables 1 and 2). The discrepancy cannot be explained by the
different size or charge of the indicated compounds; e.g.,
mannose-6-phosphate and glucose-6-phosphate are epimers. A
more reasonable explanation is that the contribution of the
translocon to the transmembrane flux is remarkable in cases
when other possibilities for membrane transport are absent (or
have a very limited capacity). Indeed, a high capacity ER
transporter has been already characterized for glucose-6-phosphate (14, 15, 19). Glutathione (2) and glucuronide (1, 4, 11)
transporters are also known, although they have been explored
only functionally. On the other hand, no transporter for mannose-6-phosphate has been even hypothesized in the ER, and
the existence and functioning of the ER UDP-glucuronic acid
transporter is controversial (1, 7).
This explanation is supported by the finding that puromycin
exerted only a slight increase even in cases when it was
effective. Effects of similar magnitude have been found in
previous studies on calcium (27, 40) and 4-methylumbelliferylglucoside permeation (18). However, it does not necessarily
mean that translocon channel plays a minor role in the permeation across the ER membrane, because the basal rate of
transport (i.e., in the absence of puromycin) observed in
microsomal vesicles might well be due to translocons unoccupied by polypeptide chains. The difference in permeability
between smooth and rough microsomal vesicles (Table 4)
seems to favor this hypothesis.
The flux of low molecular weight compounds through translocon channels can be regarded as a structural imperfection
which alters the barrier function of the ER membrane. Nevertheless, it might have a physiological function as a low-affinity,
low-capacity transporter with low selectivity. A variety of
structurally unrelated compounds are (or should be) able to
cross the ER membrane, but only a few transporters have been
reported so far (26). For example, various xenobiotics should
reach the luminal compartment of the ER to be glucuronidated.
Translocon channels may have a role in their transport. Moreover, nucleotides and nucleotide sugars are usually transported
by antiport mechanisms in the ER (6, 22). The counter ion in
these cases can be supplied by translocon. The role of the
translocon has been also hypothesized in the transport of such
a bulky anion like FAD (39) or in the permeation of exogenous
biotin derivatives (26).
In summary, the translocon channel mediates the permeation
of low molecular weight anions in the ER. The phenomenon
may play a role in the substrate supply and/or product elimination of some intraluminal enzymatic activities in the ER.
ANION TRANSPORT BY TRANSLOCON
6.
7.
8.
9.
10.
11.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
AJP-Cell Physiol • VOL
23. Johansson FI, Michalecka AM, Moller IM, and Rasmusson AG.
Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria. Biochem J 380: 193–202, 2004.
24. Johnson AE and van Waes MA. The translocon: a dynamic gateway at
the ER membrane. Annu Rev Cell Dev Biol 15: 799 – 842, 1999.
25. Latimer P. Light scattering and absorption as methods of studying cell
population parameters. Annu Rev Biophys Bioeng 11: 129 –150, 1982.
26. Le Gall S, Neuhof A, and Rapoport T. The endoplasmic reticulum
membrane is permeable to small molecules. Mol Biol Cell 15: 447– 455,
2004.
27. Lomax RB, Camello C, Van Coppenolle F, Petersen OH, and Tepikin
AV. Basal and physiological Ca2⫹ leak from the endoplasmic reticulum of
pancreatic acinar cells. Second messenger-activated channels and translocons. J Biol Chem 277: 26479 –26485, 2002.
28. Maden BE. Historical review: peptidyl transfer, the Monro era. Trends
Biochem Sci 28: 619 – 624, 2003.
29. Meissner G. Ionic permeability of isolated muscle sarcoplasmic reticulum
and liver endoplasmic reticulum vesicles. Methods Enzymol 157: 417–
437, 1988.
30. Potter MD and Nicchitta CV. Endoplasmic reticulum-bound ribosomes
reside in stable association with the translocon following termination of
protein synthesis. J Biol Chem 277: 23314 –23320, 2002.
31. Puskás F, Marcolongo P, Watkins SL, Mandl J, Allan BB, Houston P,
Burchell A, Benedetti A, and Bánhegyi G. Conformational change of the
catalytic subunit of glucose-6-phosphatase in rat liver during the fetal-toneonatal transition. J Biol Chem 274: 117–122, 1999.
32. Rajasekaran AK, Langhans-Rajasekaran SA, Gould RM, RodriguezBoulan E, and Morimoto T. A simple biochemical approach to quantitate
rough endoplasmic reticulum. Am J Physiol Cell Physiol 268: C308 –
C316, 1995.
33. Roy A and Wonderlin WF. The permeability of the endoplasmic reticulum is dynamically coupled to protein synthesis. J Biol Chem 278:
4397– 4403, 2003.
34. Seiser RM and Nicchitta CV. The fate of membrane-bound ribosomes
following the termination of protein synthesis. J Biol Chem 275: 33820 –
33827, 2000.
35. Simon SM and Blobel G. A protein-conducting channel in the endoplasmic reticulum. Cell 65: 371–380, 1991.
36. Simon SM and Blobel G. Signal peptides open protein-conducting
channels in E. coli. Cell 69: 677– 684, 1992.
37. Simon SM and Blobel G. Mechanisms of translocation of proteins across
membranes. Subcell Biochem 21: 1–15, 1993.
38. Traut RR and Monro RE. The puromycin reaction and its relation to
protein synthesis. J Mol Biol 10: 63–72, 1964.
39. Tu BP and Weissman JS. The FAD- and O2-dependent reaction cycle of
Ero1-mediated oxidative protein folding in the endoplasmic reticulum.
Mol Cell 10: 983–994, 2002.
40. Van Coppenolle F, Van den Abeele F, Slomianny C, Flourakis M,
Hesketh J, Dewailly E, and Prevarskaya N. Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores. J Cell
Sci 117: 4135– 4142, 2004.
41. Van Schaftingen E and Gerin I. The glucose-6-phosphatase system.
Biochem J 362: 513–532, 2002.
42. Zhen L, Baumann H, Novak EK, and Swank RT. The signal for
retention of the Egasyn-glucuronidase complex within the endoplasmic
reticulum. Arch Biochem Biophys 304: 402– 414, 1993.
291 • SEPTEMBER 2006 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on July 31, 2017
12.
mal fractions. Effects of inositol 1,4,5-trisphosphate and GTP. J Biol
Chem 263: 3466 –3473, 1988.
Berninsone P and Hirschberg CB. Nucleotide sugars, nucleotide sulfate,
and ATP transporters of the endoplasmic reticulum and Golgi apparatus.
Ann NY Acad Sci 842: 91–99, 1998.
Bossuyt X and Blanckaert N. Carrier-mediated transport of intact UDPglucuronic acid into the lumen of endoplasmic-reticulum-derived vesicles
from rat liver. Biochem J 302: 261–269, 1994.
Burchell A, Hume R, and Burchell B. A new microtechnique for the
analysis of the human hepatic microsomal glucose-6-phosphatase system.
Clin Chim Acta 173: 183–192, 1988.
Butler J, Spielberg SP, and Schulman JD. Reduction of disulfidecontaining amines amino acids, and small peptides. Anal Biochem 75:
674 – 675, 1976.
Csala M, Bánhegyi G, Braun L, Szirmai R, Burchell A, Burchell B,
Benedetti A, and Mandl J. ␤-Glucuronidase latency in isolated murine
hepatocytes. Biochem Pharmacol 59: 801– 805, 2000.
Csala M, Staines AG, Bánhegyi G, Mandl J, Coughtrie MW, and
Burchell B. Evidence for multiple glucuronide transporters in rat liver
microsomes. Biochem Pharmacol 68: 1353–1362, 2004.
Czosnek HH, de Groot N, and Hochberg AA. The comparison of rat
liver rough endoplasmic reticulum membrane proteins before and after in
vitro removal of its bound ribosomes. Mol Biol Rep 2: 113–118, 1975.
Fulceri R, Bánhegyi G, Gamberucci A, Giunti R, Mandl J, and
Benedetti A. Evidence for the intraluminal positioning of p-nitrophenol
UDP-glucuronosyltransferase activity in rat liver microsomal vesicles.
Arch Biochem Biophys 309: 43– 46, 1994.
Galli L, Orrico A, Marcolongo P, Fulceri R, Burchell A, Melis D,
Parini R, Gatti R, Lam C, Benedetti A, and Sorrentino V. Mutations in
the glucose-6-phosphate transporter (G6PT) gene in patients with glycogen storage diseases type 1b and 1c. FEBS Lett 459: 255–258, 1999.
Gerin I, Veiga-da-Cunha M, Noel G, and Van Schaftingen E. Structure
of the gene mutated in glycogen storage disease type Ib. Gene 227:
189 –195, 1999.
Haigh NG and Johnson AE. A new role for BiP: closing the aqueous
translocon pore during protein integration into the ER membrane. J Cell
Biol 156: 261–270, 2002.
Hamman BD, Hendershot LM, and Johnson AE. BiP maintains the
permeability barrier of the ER membrane by sealing the lumenal end of the
translocon pore before and early in translocation. Cell 92: 747–758, 1998.
Heritage D and Wonderlin WF. Translocon pores in the endoplasmic
reticulum are permeable to a neutral, polar molecule. J Biol Chem 276:
22655–22662, 2001.
Hiraiwa H, Pan CJ, Lin B, Moses SW, and Chou JY. Inactivation of the
glucose 6-phosphate transporter causes glycogen storage disease type 1b.
J Biol Chem 274: 5532–5536, 1999.
Hirschberg CB, Robbins PW, and Abeijon C. Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and
Golgi apparatus. Annu Rev Biochem 67: 49 – 69, 1998.
Ioannou M, Coutsogeorgopoulos C, and Synetos D. Kinetics of inhibition of rabbit reticulocyte peptidyltransferase by anisomycin and sparsomycin. Mol Pharmacol 53: 1089 –1096, 1998.
Ishida N and Kawakita M. Molecular physiology and pathology of the
nucleotide sugar transporter family (SLC35). Pflügers Arch 447: 768 –775,
2004.
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