Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France
Large, free polymannose oligosaccharides
generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo
processing by a cytosolic endo H-like enzyme and a
mannosidase to yield the linear isomer of Man5GlcNAc (Man[1-2]Man[
1-2]Man[
1-3][Man
1-6]Man[
14]GlcNAc). Here we have examined the fate of these
partially trimmed oligosaccharides in intact HepG2
cells. Subsequent to pulse-chase incubations with d-[2-
3H]mannose followed by permeabilization of cells with
streptolysin O free oligosaccharides were isolated from
the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that
total cellular free oligosaccharides are lost from HepG2
cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized
total cellular free oligosaccharides and enabled us to
demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound
compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells
were treated with either 100 µM swainsonine, which
provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or
agents known to reduce cellular ATP levels which
lead to the accumulation of the linear isomer of
Man5GlcNAc in the cytosol. Subcellular fractionation
studies on Percoll density gradients revealed that the
cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.
The glycosylation of proteins with N-linked carbohydrate in the endoplasmic reticulum is a common
and important posttranslational modification. Surprisingly, this process, accomplished by the transfer of a
polymannose-type oligosaccharide from a lipid carrier (dolichol) onto polypeptide (Kornfeld and Kornfeld, 1985 It has been known for some years that the cytosol contains both an endo H-like enzyme (Pierce et al., 1979 Here we report on the fate of this cytosolic free oligosaccharide in intact HepG2 cells. It is shown that partially trimmed cytosolic oligosaccharides are translocated
into a membrane bound compartment by a nonautophagic
process that requires energy. Subcellular fractionation of
HepG2 cell homogenates on Percoll density gradients revealed that cytosolic oligosaccharides are ultimately
degraded in a compartment that cosediments with lysosomes. Along with a previous report describing the transport of free oligosaccharides from the lumen of the ER
into the cytosol (Moore et al., 1995 Culture and Radiolabeling of Cells
HepG2 cells were cultivated in RPMI 1640 (GIBCO BRL, Paisley, UK)
containing 10% FCS (GIBCO) as previously described (Moore and Spiro,
1990 Permeabilization of Cells
At the end of pulse-chase experiments, cells were released from tissue
culture flasks with trypsin/EDTA and washed twice in 1.0 ml of permeabilization buffer: 250 mM mannitol, 5 mM Hepes (pH 7.3), 2 mM EGTA, 1 mM
CaCl2, 2 mM MgCl2. Cells were then incubated at 4°C for 20 min in 0.5 ml
of the permeabilization buffer containing 1 U/ml streptolysin O (Wellcome Diagnostics, Dartford, UK). Cells were recovered by centrifugation
and the supernatant was kept. Subsequent to washing the cells twice with
0.5 ml permeabilization buffer at 4°C, they were incubated with 0.5 ml
prewarmed permeabilization buffer (37°C) for 5 min and the permeabilized cells were then recovered by centrifugation to yield the membranebound compartment (MBC)1 fraction. The final supernatant was pooled
with the SLO-containing supernatant and the two subsequent permeabilization buffer washes to yield 2 ml of the cytosolic compartment fraction
(Cytosol).
Preparation of Free Oligosaccharides from Cytosolic
and MBC Fractions of HepG2 Cells
Neutral free oligosaccharides were prepared from the cytosol and MBC
fractions as previously described (Moore and Spiro, 1994 Structural Analysis of Man5GlcNAc Oligosaccharides
After resolution of free oligosaccharides by thin layer chromatography
components of interest were eluted from the chromatography plates with
water and passed over coupled columns of AG 50 (H+ form) and AG 1 (acetate form). Nonretained neutral components were dried and subjected to two mannosidase treatments. One aliquot was treated with 1 U
Jack bean Percoll Density Gradient Fractionation
HepG2 cells were washed three times with ice-cold PBS containing 1 mM
CaCl2, 1 mM MgCl2 and once with ice-cold subcellular fractionation
buffer (SFB), 250 mM sucrose, 20 mM Hepes, 1 mM EDTA, pH 7.2. The
cells were scraped from tissue culture flasks in 5 ml SFB and cellular protein was assayed using a bicinchoninic acid protein assay kit (Sigma
Chemical Co.). The cell pellet obtained after centrifugation at 600 g for 10 min was resuspended in SFB (1.5 mg/ml protein) and placed on ice for 15 min. Cell homogenization was carried out using a tight-fitting Dounce homogenizer (30 passages). After centrifuging the homogenate at 600 g for
10 min, the supernatant was removed and kept on ice, and the pellet was
resuspended with SFB and rehomogenized and centrifuged as above.
Pooled supernatants were adjusted to 5 ml with SFB and 3 ml of an 80%
Percoll solution was added (Rijnboutt et al., 1992
HRP Uptake
HepG2 cells were washed with MEMH (MEM containing 1 mM sodium
pyruvate, 1 mM l-glutamine and 20 mM Hepes/NaOH, pH 7.2) and HRP
uptake was performed in MEMH containing 5 mg/ml HRP (Sigma Chemical Co.) for 5 or 15 min at 37°C (van Weert et al., 1995). Extracellular
HRP was then removed by washing the cells with ice-cold MEMH over a
period of 10 min, followed by either a 5-min or a 2-h chase period, corresponding to the 5 or 15 min pulse times, respectively (van Weert et al.,
1995). HRP-loaded cells were then washed and homogenized as described
above.
Enzymatic Assays
HRP activity was assayed in 50 mM phosphate buffer (pH 5.0) containing
0.1% Triton X-100, using 83 µg/ml O-dianisidine and 1% H2O2 as substrates (van Weert et al., 1995). The reaction was performed for 5 min at
room temperature in the dark; absorbance was measured at 460 nm.
Lysosomal NADPH cytochrome c reductase activity was measured as previously
described in a 50 mM phosphate, 0.1 mM EDTA buffer, pH 7.7, using 1 mg/ml NADPH and 25 µg/ml cytochrome c as substrates. Absorbance increases were measured at 550 nm over a 3-min period.
High pH Anion Exchange Chromatography
High pH anion exchange chromatography (HPAEC) was carried out on a
Dionex apparatus as previously described (Townsend et al., 1991 Free Oligosaccharides Appear Rapidly in the Cytosol
of HepG2 Cells during Glycoprotein Biosynthesis and
Are Then Cleared from This Compartment
In a previous study we have shown that free oligosaccharides generated from oligosaccharide-lipid, bearing the di-
N-acetylchitobiose moiety (OS-GN2), are rapidly transported out of the ER into the cytosol (Moore et al., 1995
The Clearance of Free Oligosaccharides
from the Cytosol Is Not Dependent upon Vacuolar
H+/ATPase Activity
CCM A (Woo et al., 1992 These results show that CCM A has only small effects
on the appearance and decay of radioactivity associated
with free oligosaccharides in the cytosol of HepG2 cells
but, in contrast, this reagent provokes a marked accumulation of free oligosaccharides associated with an MBC.
The Isomeric Structure of the Man5GlcNAc
Isolated from MBCs of CCM A-treated HepG2 Cells
Is Consistent with Its Cytosolic Origin
To evaluate the possibility that the CCM A-provoked accumulation of free oligosaccharides associated with an
MBC is related to the loss of these components from the
cytosol, we next investigated the isomeric configuration of
the Man5GlcNAc associated with the MBCs. The cytosol is
known to contain an endo H-like enzyme and an The Loss of Free Oligosaccharides from the
Cytosol Can Be Accounted for Both Quantitatively and
Kinetically by Their Recovery within an MBC of CCM
A-treated HepG2 Cells
The ability of CCM A to block the degradation of MBCassociated free polymannose type oligosaccharides has enabled us to further establish the hypothesis that there is a
cytosol-to-MBC translocation of free oligosaccharides. Accordingly, we have verified that the loss of free oligosaccharides from the cytosol could be quantitatively and kinetically accounted for by their reappearance in an MBC. Quantitation of the free oligosaccharides generated during
the control and CCM A pulse-chase incubations is shown
in Fig. 3. In the control incubations there is a loss of total
free oligosaccharide from cells such that after 8 h of chase
only 25% of the cytosolic free oligosaccharides remain.
However in the presence of CCM A the total quantity of
free oligosaccharide remains approximately constant between 1 and 8 h of chase and the loss of free oligosaccharides from the cytosol can be accounted for, both quantitatively and kinetically, by their recovery from the MBCs
(Fig. 3).
Next, we wanted to know whether the free oligosaccharides associated with the MBCs of CCM A-treated HepG2
cells are located within the MBC or are merely associated
with the cytosolic face of its delineating membrane. This
problem was addressed by performing a pulse-chase incubation in the presence of CCM A as described in Fig. 1.
After 6 h of chase the cells were first permeabilized with
SLO to release the remaining cytosolic oligosaccharides
and then repermeabilized with saponin. Results indicated
that >90% of the free oligosaccharides associated with the
MBCs of SLO-permeabilized cells could be released upon
their repermeabilization with saponin (results not shown).
Identical results were obtained if the saponin treatment
was substituted by freeze-thawing (not shown). These results confirm the hypothesis that in CCM A-treated
HepG2 cells there is a translocation of free oligosaccharides from the cytosolic compartment into the lumen of an
MBC.
Inhibition of Cytosolic Processing of Free
Polymannose-type Oligosaccharides Affects Their
Subcellular Trafficking
We then wanted to know if the structure of cytosolic-free
oligosaccharides play a role in their transfer into an MBC.
To achieve this the total cellular molar equivalent of each
free oligosaccharide was calculated after 1, 2, and 4 h of
chase in the presence of CCM A as shown in the upper
part of Fig. 4. The percentage of each oligosaccharide occurring in the MBCs was then computed and displayed in
the lower portion of Fig. 4. Irrespective of the total cellular
quantity of Man9-8GlcNAc ~17% of these two oligosaccharides are recovered from the MBC after 1, 2, and 4 h of
chase. Because the permeabilization procedure used in
these studies leads to the release of ~80% of cellular lactate dehydrogenase (results not shown), the small percentage of Man9-8GlcNAc occurring in the MBCs during the
chase incubations may represent free oligosaccharides in
nonpermeabilized cells. However, as the chase progresses
there is a steady increase in the proportion of free Man7-5-
GlcNAc oligosaccharides occurring in the MBCs. These
results suggest that oligosaccharides bearing 8 or 9 residues of mannose might be poorly transferred into a MBC.
To gain more insight into the mechanism of the transfer of
free oligosaccharides from the cytosol to a MBC, we examined the effect of inhibition of the cytosolic mannosidase on the transfer of cytosolic oligosaccharides into the
MBC. If, as suggested above, large free oligosaccharides
bearing 8 or 9 residues of mannose are poorly transferred
from the cytosol into an MBC, then an inhibitor of the cytosolic mannosidase would be expected to slow down the
transfer of the larger free polymannose-type oligosaccharides from the cytosol into an MBC of HepG2 cells. At
high concentrations swainsonine (SW), a nonspecific mannosidase inhibitor (Elbein et al., 1981
The Golgi Apparatus Does Not Play a Major Role in the
Trafficking of Free Oligosaccharides in HepG2 Cells
Taken together the results presented so far provide evidence to show that most free oligosaccharides follow a
trafficking pathway from the ER into the cytosol and then
into an MBC possessing a mannosidase with a low pH optimum. However, the appearance of the branched isomer
of Man5GlcNAc in the MBCs of SW-treated cells suggested that there may also exist a vesicular trafficking
pathway for free oligosaccharides involving the Golgi complex. Furthermore, as CCM A is known to affect the
movement of glycoproteins through the Golgi complex
(Yilla et al., 1993
Inhibitors of Macroautophagy Do Not Affect the
Clearance of Free Oligosaccharides from the Cytosol of
HepG2 Cells
Having demonstrated that the bulk of free oligosaccharides are transferred from the cytosol into a degradative
organelle, we went on to investigate the mechanism of this
translocation process. Theoretically macroautophagy could
be responsible for the clearance of oligosaccharides from
the cytosol. To test this possibility we have performed
chase incubations in the presence of well known inhibitors
of macroautophagy. Pulse-chase experiments were performed as described in Fig. 1 except that the inhibitors were added to the chase incubations 45 min after the onset
of the chase period. 3-MA inhibits the sequestration step
of the autophagic process (Seglen and Gordon, 1982
The Transfer of Free Oligosaccharides from the Cytosol
into an MBC Requires Energy
To further investigate the nature of the cytosol-to-MBC
translocation of free oligosaccharides in HepG2 cells, we
have investigated the energy dependence of this process.
For this purpose we have performed chase incubations in
the presence of reagents known to deplete ATP levels in
cultured cells (Krijnse-Locker et al., 1994
Subcellular Fractionation Reveals That the
Pseudolinear Isomer of Man5GlcNAc Is Degraded
in Lysosomes
We have examined the identity of the MBC in which cytosolically generated free oligosaccharides are degraded by
subcellular fractionation. As in the presence of CCM A,
the linear isomer of Man5GlcNAc is stabilized in an MBC
of permeabilized HepG2 cells we reasoned that in the absence of this drug free oligosaccharides are degraded
within a subcellular compartment possessing a vacuolar H+/ATPase and an Due to difficulties in interpreting these results we have
used 0.1 µM SW to provoke an accumulation of free oligosaccharides in the MBC of HepG2 cells before subcellular fractionation. As described above we have found that,
at a concentration of 0.1 µM, SW mimics the effects of
CCM A on the subcellular trafficking of free oligosaccharides in HepG2 cells. Treatment of HepG2 cells with 0.1 µM SW for 4 h did not dramatically affect the distributions of the lysosomal, Golgi, or endoplasmic reticulum marker
enzymes along the Percoll gradient (compare the top
panel of Fig. 10 with control fractionations in Fig. 9 A). To
identify endosomal and lysosomal compartments, HepG2
cells, treated for 4 h with 0.1 µM SW, were allowed to endocytose a brief pulse of the fluid phase marker, HRP, and
then chased for various times (van Weert et al., 1995). Cellular homogenates were then fractionated on Percoll density gradients as shown in Fig. 10 (bottom). Results show
that, after the pulse, HRP is found uniquely in a region of
the gradient (see top, fraction II), in which are found light
membranes, including the endoplasmic reticulum and
Golgi apparatus. After 4 h of chase the HRP is now mainly
found at the bottom of the gradient, a region shown to
contain the lysosomes (see top, fraction IV). These results are consistent with the transfer of the fluid phase marker
from the endosomal compartment to the lysosomal compartment as previously described in HepG2 cells (van Weert
et al., 1995). Cells treated with 0.1 µM SW were then pulse
radiolabeled, chased for 1 and 4 h, homogenized, and then
subjected to subcellular fractionation on Percoll density
gradients. Fig. 11 A shows that after the pulse free oligosaccharides (OS-GN2, results not shown) are localized to a region of the gradient corresponding to the endoplasmic reticulum/Golgi region of the gradient, after 4 h of
chase free oligosaccharides (mainly Man7-5GlcNAc, results
not shown) now colocalize with the lysosomal marker enzyme
Permeabilization of the plasma membrane of HepG2 cells
subsequent to pulse-chase incubations has enabled us to
evaluate the hypothesis that cytosolic-free oligosaccharides are sequestered into and degraded by lysosomes.
Short chase incubations revealed that free oligosaccharides rapidly appear in the cytosol at a time during which
there is a loss of these components from the MBCs (Fig.
1, A and B). This observation can be accounted for by the
previously observed rapid translocation of large, free polymannose-type oligosaccharides out of the ER into the cytosol (Moore et al., 1995 After Trimming in the Cytosol-free
Oligosaccharides Are Translocated into Lysosomes
Where They Are Degraded
Subcellular fractionation of HepG2 cells chased in the
presence of 0.1 µM SW for 1 and 4 h indicated that the distribution of free oligosaccharides along Percoll gradients
was the same as that observed for the lysosomal marker
enzyme How Are Free Oligosaccharides Cleared from
the Cytosol?
Examining the kinetics of loss of free oligosaccharides
from cytosol yields useful information concerning the
mechanism of this translocation process. First, as the cytoplasm of cells is continually sequestered by vesicles and
delivered to lysosomes by macroautophagy, we wondered
whether or not this bulk sequestration of the cytosol could
account for the delivery of free cytosolic oligosaccharides
to the lysosome. It has been shown that by starving hepatocytes of serum, autophagic sequestration can be stimulated 10-fold and under these conditions only 4% of the
cytoplasm can be sequestered per hour (Kopitz et al.,
1990 What Are the Consequences of the Sequestration of
Cytosolic-free Oligosaccharides in Lysosomes?
The results we have obtained with HepG2 cells throw light
on a problem that has perplexed researchers investigating
the genetic disorder, An intriguing question can now to be addressed: Why
has the cell developed such an elaborate trafficking pathway for the degradation of free oligosaccharides generated
in the lumen of the ER when two vesicular pathways already exist between the ER and lysosome? One explanation for this is that free oligosaccharides must be rapidly
segregated from their N-linked counterparts in the ER in
order to minimize their interference with the folding and
trafficking of glycoproteins, a process now thought to involve lectins situated along the secretory pathway (Fiedler
and Simmons, 1995 In conclusion our results show that after their rapid
appearance in the cytosol during glycoprotein biosynthesis
free oligosaccharides are trimmed by the cytosolic mannosidase and transferred into lysosomes by an energy-
dependent mechanism. Our results show that the lysosome
is the site for the final degradation of these oligosaccharides and suggest that the lysosomal membrane is itself involved in the uptake of free polymannose-type oligosaccharides from the cytosol.
),
is accompanied by the release of free polymannose-type
oligosaccharides into the lumen of the ER (Anumula and
Spiro, 1983
; Cacan et al., 1987
). As large amounts of free
oligosaccharides are generated in this way an understanding of the fate of this material became important. It was
initially thought that free oligosaccharides generated in
the lumen of the ER might be exported from the cell by
vesicular transport as a consequence of the effect of bulk flow (Wieland et al., 1987
). In fact this was found not to be the case as free oligosaccharides were not recovered from
the incubation media of cultured HepG2 cells (Moore and
Spiro, 1990
) but detected in the cytosol (Moore and Spiro,
1994
). More recently free polymannose-type oligosaccharides bearing the terminal reducing di-N-acetylchitobiose
moiety have been shown to be transported out of the ER
into the cytosol in permeabilized HepG2 cells (Moore et al.,
1995
). In addition to the transfer of free oligosaccharides from the lumen of the ER into the cytosol, there is now evidence to suggest that some free oligosaccharides may be
generated in the cytosol by either the release of cytosolically disposed oligosaccharides from dolichol (Kmiécik et al.,
1995
), or by the degradation of glycoproteins, initiated by
a cytosolic N-glycanase (Suzuki et al., 1994
), that have been
translocated out of the ER into the cytosol (Wiertz et al.,
1996
). These reports highlight the crucial role that the cytosol plays in the processing and perhaps generation of
free oligosaccharides which are generated during the biosynthesis and quality control of glycoproteins. What then
is the fate of these free cytosolic oligosaccharides?
) and
an
-mannosidase (Shoup and Touster, 1976
; Tulsiani and
Touster, 1987
). In vitro experiments with preparations of
the cytosolic
-mannosidase have revealed it to possess two
notable features, firstly it is inactive towards large polymannose-type oligosaccharides bearing the di-N-acetylchitobiose moiety at their reducing termini (Oku and Hase, 1991
)
and secondly its limit digest product is the linear isomer of
Man5GlcNAc: Man(
1-2)Man(
1-2)Man(
1-3)(Man[
16])Man(
1-4)GlcNAc; Tulsiani and Touster, 1987
; Oku and
Hase, 1991
). In intact cells cytosolic-free oligosaccharides
generated during the biosynthesis of glycoproteins are apparently subjected to the actions of these two cytosolic enzymes to yield the linear isomer of Man5GlcNAc (Moore
and Spiro, 1994
).
), this report describes a
novel trafficking pathway for free oligosaccharides that
links the endoplasmic reticulum to the lysosome via the
cytosol.
Materials and Methods
). Cells were pulse radiolabeled with 40 µCi d-[2-3H]mannose (20 Ci/
mmol; Amersham, Slough, UK) for 20 min in 0.5 ml glucose-free Dulbecco's modified Eagle Medium (GIBCO BRL) supplemented with 5%
dialyzed FCS, 2 mM glutamine, 5 mM fucose, and 1 mM sodium pyruvate.
Cells were chased in complete growth medium containing 5 mM fucose
and 5 mM mannose. When pulse-chase studies were performed in the
presence of swainsonine (Sigma Chemical Co., St. Louis, MO) and concanamycin A (Ciba-Geigy, Switzerland), the cells were preincubated with
these inhibitors for 1 h before the onset of radiolabeling and were added
to both the pulse and chase media at the appropriate concentrations. Before subcellular fractionation studies cells were pulse radiolabeled for 20 min with 200 µCi d-[2-3H]mannose and chased as described above. Other
drugs including 3-methyladenine, asparagine, deoxyglucose, sodium azide,
and oligomycin (all from Sigma Chemical Co.) were included in the chase
media only and were added 45 min after the onset of the chase incubations, whereas leupeptin (Sigma Chemical Co.) was added at the beginning of each chase period.
). Briefly, the
pellet of permeabilized cells (MBC) was extracted with chloroform/methanol/125 mM Hepes (pH 7.2) containing, 4 mM MgCl2, 3:2:1, and after vigorous shaking the upper methanolic phase was recovered, dried, and redissolved in water. This material and the cytosolic fractions were desalted
on combined columns of AG 1-X2 (acetate form) and AG 50-X2 (H+
form), unbound neutral material was then loaded onto columns of charcoal, which were then washed with water before elution of oligosaccharide
material from the charcoal with 30% ethanol. Free oligosaccharides were
analyzed on plastic thin layer chromatography plates coated with silica
(Merck, Darmstadt, Germany) which were developed in n-propanol/acetic acid/water (3:2:1) for 12 h. Resolved components were visualized by
fluorography.
-mannosidase (Sigma Chemical Co.) overnight at 37°C in 40 mM
sodium acetate, pH 4.5. Another aliquot was digested overnight at 37°C
with 5 µU
1-2 mannosidase (Oxford Glycosystems, Abingdon, UK) in
100 mM sodium acetate buffer, pH 5.0. The digestion products were then
desalted as described above, concentrated, and resolved by thin layer
chromatography on plastic sheets coated with cellulose (0.1 mm thickness;
Merck). Chromatographs were developed in pyridine/ethyl acetate/water/
acetic acid 5:5:3:1 for 10 h, and after drying resolved components were visualized by fluorography. Quantitation of the resolved products was
achieved by their elution from the cellulose plates and assaying radioactive components by scintillation counting.
). The gradient was
formed by centrifugation for 35 min at 92,570 gAv. (Rijnboutt et al.,
1992
). 400-µl fractions were collected from the top of the tube with a needle mounted on a syringe. To isolate free oligosaccharides from the Percoll gradient, fractions were pooled (see Fig. 9), diluted in SFB, and after
centrifugation for 90 min at 100,000 gAv organelles were recovered separately from the Percoll pellet. Free oligosaccharides were prepared from
organelle fractions as described above, except that before ion-exchange
chromatography, sucrose was eliminated from the samples by Biogel P2
gel filtration.
Fig. 9.
CCM A affects the
distribution of a lysosomal
marker enzyme on Percoll
density gradients. (A) Control cells (open circles) or
cells treated with CCM A for
4 h were homogenized and
fractionated on Percoll density gradients as described in
Materials and Methods. The
sedimentation position of lysosomes, the Golgi apparatus, and ER were identified
by performing assays for
-d-hexosaminidase (
-
HEX),
-1, 4-galactosyltransferase (GAL T'ASE),
and NADPH cytochrome
C reductase (NADPH
RED'ASE), respectively. (B)
CCM A-treated cells were
pulse radiolabeled (PULSE)
and chased (CHASE) in the
presence of CCM A for 4 h
before subcellular fractionation as described above. After collecting the gradient in
four fractions (I-IV) as
shown in the upper panel of
A, organelles were separated from Percoll and cytosol by centrifugation as described in Materials and Methods. Free oligosaccharides
were isolated from the resulting organelles and assayed by scintillation counting.
[View Larger Version of this Image (36K GIF file)]
-d-hexosaminidase activity was measured using p-nitrophenyl N-acetylglucosamine as described previously (Opheim and Touster,
1977
).
1, 4 Galactosyltransferase was assayed by the method of Barker
et al. (1972)
.
). Components were eluted at 1 ml/min with buffer A for 10 min followed by a linear gradient of 0-10% buffer B over 25 min (buffer A: 50 mM NaOH; buffer
B: 500 mM sodium acetate in buffer A). Column effluent was monitored
for radioactive components with a Flow Scintillation Analyzer Radiomatic
Flow-one/
(Packard, Instrument Company, Meriden, CT) using a scintillation fluid (UltimaFlow one AP, Packard) flow rate of 2 ml/min. Standard
oligosaccharides, prepared as previously described (Michalski et al., 1990
;
Haeuw et al., 1991
) were monitored by pulsed electrochemical detection.
Results
).
This process was observed in vitro upon addition of ATP
to permeabilized HepG2 cells. In the present report we
have followed the subcellular trafficking of free polymannose oligosaccharides in vivo by permeabilizing the plasma
membrane of [3H]mannose-radiolabeled HepG2 cells with
SLO. After centrifugation of permeabilized cells, free oligosaccharides were isolated from the supernatant (cytosolic compartment) and the residual cell pellet, containing
intact intracellular organelles (membrane bound compartments; MBCs). Fig. 1 A shows the results of such an experiment. During the pulse the MBC contains free oligosaccharides (OS-GN2; Fig. 1, open arrowheads) whereas the
cytosol contains the same components and in addition
their counterparts bearing a single residue of N-acetylglucosamine at their reducing termini (OS-GN1). Between
the pulse and 1-h chase period there is a loss of OS-GN2 from the MBC and an increase in free oligosaccharides in
the cytosolic compartment. These observations represent
the previously described ER-to-cytosol transport process
(Moore et al., 1995
). After transport out of the ER, OSGN2 are subject to a cytosolic endo H-like activity to yield
their OS-GN1 counterparts. OS-GN1 are now potential
substrates for the cytosolic mannosidase which trims these
components down to a limit digest product; the linear isomer of Man5GlcNAc (Moore and Spiro, 1994
; see Fig. 2 B
for representation of this structure). However, as can be
seen in Fig. 1 A, free oligosaccharides neither accumulate
in the cytosol nor reappear in an MBC. We reasoned that
if cytosolic oligosaccharides are translocated into a degradative compartment they might be difficult to detect due
to their rapid hydrolysis into free mannose and N-acetylglucosamine. Because the lysosome is the most likely compartment in which cytosolic oligosaccharides might be degraded we chose to perform pulse-chase experiments in
the presence of a vacuolar H+/ATPase inhibitor.
Fig. 1.
The effect of CCM
A on the generation and fate
of free oligosaccharides in
HepG2 cells. Control (A) or
CCM A-treated (B) HepG2 cells were pulse radiolabeled
with d-[2-3H]mannose for 20 min, and then chased for the
indicated times. Cells were
then cooled, placed in suspension, and permeabilized with
SLO as described in Materials
and Methods to yield fractions corresponding to the cytosol
(CYTOSOL) and membrane-
bound compartments (MBCs).
Free oligosaccharides were
prepared from each of these fractions and then resolved
by thin layer chromatography,
on silica-coated plates. The
abbreviations associated with
the solid arrowheads are:
G1M9, Glc1Man9GlcNAc; M9,
Man9 GlcNAc; M8, Man8GlcNAc; M7, Man7GlcNAc;
M6, Man6GlcNAc; M5, Man5GlcNAc; M4, Man4GlcNAc;
M3, Man3GlcNAc. Those associated with the open arrowheads are: G1M9, Glc1Man9
GlcNAc2; M9, Man9GlcNAc2;
M8, Man8GlcNAc2.
[View Larger Version of this Image (51K GIF file)]
Fig. 2.
The linear isomer of Man5GlcNAc accumulates in a
membrane-bound compartment of CCM A-treated HepG2 cells.
HepG2 cells were pulse radiolabeled and chased for 4 h in the
presence of CCM A as described in Materials and Methods. The
cells were then permeabilized with SLO and oligosaccharides
were purified from the resulting MBC as previously described.
The component migrating as Man5GlcNAc was recovered and aliquots of this oligosaccharide were then digested with either jack
bean -mannosidase (JACK BEAN) or an
-1, 2 mannosidase (
-1, 2). After desalting, the digestion products were resolved by
thin layer chromatography on a cellulose-coated plate. Subsequent to visualization of resolved components by fluorography
they were eluted from the chromatography plate and assayed
by scintillation counting. The molar equivalents of the resolved
components were calculated and, after summing, the percentage molar distribution of the digestion products was calculated.
The cytosolic mannosidase generates an isomer (LINEAR) of
Man5GlcNAc that contains two
-1, 2-linked mannose residues
whereas Golgi mannosidase I leads to the formation of a
Man5GlcNAc isomer (BRANCHED) devoid of
-1, 2-linked
mannose residues. The abbreviations are as follows: M5GN, Man5GlcNAc; M4GN, Man4GlcNAc; M3GN, Man3GlcNAc;
M2GN, Man2GlcNAc; MGN, ManGlcNAc; M, Mannose.
[View Larger Version of this Image (29K GIF file)]
) is a vacuolar H+/ATPase which
has been shown to abolish acidification of lysosomes without affecting cellular ATP levels (Woo et al., 1992
: Kataoka et al., 1995
), we reasoned that if free oligosaccharides
are translocated into an acidic MBC from the cytosol this
reagent may either inhibit their access to, or, failing that,
block their degradation within an acidic compartment.
Free oligosaccharides isolated from the cytosol and MBCs
of cells from a pulse-chase experiment performed in the
presence of CCM A are shown in Fig. 1 B, results show
that when compared to the control experiment (Fig. 1 A),
this reagent had very little effect on early events (0-1 h)
during the pulse-chase experiment and we conclude from
this that the generation of free oligosaccharides in the ER
and their transport out of this compartment into the cytosol is unaffected by CCM A. In contrast Fig. 1 B shows
that, after 1 h of chase, CCM A provokes a steady accumulation of free oligosaccharides bearing predominantly 7-4 residues of mannose, in an MBC. Although the effect of
CCM A is most marked with respect to oligosaccharides
recovered from the MBCs we noted systematically, after
long chase periods, that this reagent also causes a small but
significant accumulation of free oligosaccharide material
in the cytosolic compartment. We also observed, after 8 h
of chase in the presence of CCM A, that 7.9% of total free oligosaccharides produced by HepG2 cells could be recovered from the incubation medium, and after 20 h of chase
this figure rose to 18.0%. As the quantity of free oligosaccharides recovered from the incubation media of CCM
A-treated cells represented only a small fraction of total
cellular free oligosaccharides observed during the time frame
of our experiments the contribution made by these components to the quantitative aspects of our studies have not
been taken into account.
-mannosidase which together process the large oligosaccharides that are transported out of the ER into the cytosol to yield
the limit digest product Man5GlcNAc (Fig. 2, LINEAR)
(Moore and Spiro, 1994
). Although products closely related to CCM A are known to inhibit the degradation of
proteins in the lysosomes of certain cell lines (Yoshimori
et al., 1991
), we could not rule out the possibility that in
our hands CCM A caused the accumulation of partially
degraded polymannose- or hybrid-type oligosaccharides derived from incomplete lysosomal glycoprotein degradation. If this were the case, then any resulting free
Man5GlcNAc should have a structure consistent with its
passage through the Golgi apparatus while N-linked to a
protein. Accordingly, the Man5GlcNAc isolated from
MBCs of CCM A-treated HepG2 cells would be expected to be the branched isomer of this oligosaccharide (Fig. 2,
BRANCHED) (Kornfeld and Kornfeld, 1985
). Thus transport of this latter component into an MBC of CCM A-treated
HepG2 cells would lead to the accumulation of an isomer
of Man5GlcNAc different from that expected from the limit
digest product of the cytosolic mannosidase. To distinguish between the two possible origins of the Man5GlcNAc observed to accumulate in the presence of CCM A, we have
isolated this component from the MBCs and subjected it
to digestion with a nonspecific
-mannosidase (Jack bean)
and an
-1, 2 mannosidase (Amano and Kobata, 1986
) as
shown in Fig. 2. The linear isomer of Man5GlcNAc has
two
-1, 2 linked mannose residues whereas its branched
counterpart contains no such linkages. Results show that
the structure isolated from the MBCs of CCM A-treated
HepG2 cells is sensitive to the
-1, 2 mannosidase yielding
free mannose and the tetrasaccharide Man3GlcNAc. Jack
bean
-mannosidase treatment of the Man5GlcNAc yielded free mannose and the disaccharide Man
-1, 4 GlcNAc, in the ratio 4:1, indicating that this component
possessed a single terminal reducing N-acetylglucosamine
moiety and that all the
-linked mannose residues were
accessible to an exomannosidase. Similar treatment of the
Man4GlcNAc isolated from the MBCs of CCM A-treated
HepG2 cells revealed it also to be sensitive to the
-1, 2 mannosidase yielding the digest products Man3GlcNAc and mannose (results not shown). These results clearly demonstrate that the isomeric configurations of the free oligosaccharides associated with the MBCs of CCM A-treated
HepG2 cells are compatible with their having been generated by the cytosolic mannosidase and not as a consequence of incomplete degradation of free oligosaccharides derived from lysosomal glycoprotein catabolism.
Fig. 3.
Quantitation of the transfer of free oligosaccharides
from the cytosol into a membrane-bound compartment of CCM
A-treated HepG2 cells. Cells were pulse-radiolabeled and chased
in either the absence (CONTROL) or presence (CCM A) of
CCM A. After isolation from both the cytosolic and membranebound compartments free oligosaccharides were resolved by thin
layer chromatography as described in Fig. 1. Subsequent to fluorography each of the resolved components were quantitated by
densitometry. The molar equivalent (MEq.) of any given oligosaccharide was then calculated by dividing its estimated quantity
by the number of mannose residues the component contained.
Molar equivalents of all cytosolic (open circles) oligosaccharides were summed and the same procedure was applied to free oligosaccharides derived from the MBCs (closed circles). The open
triangles represent the sum of the molar equivalents of the cytosolic and MBC components. The values reported in this figure
were calculated from data obtained from two pulse-chase experiments.
[View Larger Version of this Image (23K GIF file)]
), inhibits the cytosolic
-mannosidase (Tulsiani and Touster, 1987
) in addition to Golgi mannosidase II and lysosomal mannosidases.
Accordingly, HepG2 cells were pulse radiolabeled and
chased in the presence of 100 µM SW, and, subsequent to
permeabilization with SLO, free oligosaccharides were
prepared from both the MBCs and the cytosol as described for Fig. 1. Fig. 5 A demonstrates that after 4 h of
chase SW causes an inhibition of the loss of radioactivity
associated with free oligosaccharide material from the cytosolic compartment of HepG2 cells. Furthermore thin
layer chromatography of the oligosaccharides recovered
from the cytosolic compartment of HepG2 cells chased for
4 h in the presence of 100 µM SW revealed that
Man9GlcNAc and Man8GlcNAc oligosaccharides are stabilized in the cytosol for up to 4 h and that there is little
transfer of these components into the MBC. Although Fig.
5 B shows that free oligosaccharides bearing 9 and 8 residues of mannose are stabilized in the cytosolic compartment of SW-treated cells, we observed (Fig. 5 C) a small
accumulation of Man5GlcNAc in the MBCs derived from
these cells. As described above in addition to inhibiting
the cytosolic mannosidase SW is also known to inhibit
Golgi mannosidase II which leads to the production of glycoproteins bearing hybrid-type oligosaccharide chains (Elbein et al., 1981
). Hybrid-type oligosaccharide chains possess a core structure which contains the branched
Man5GlcNAc moiety (Fig. 2), now, if such glycoproteins,
or free oligosaccharides, are transported from the Golgi
complex to lysosomes, whose mannosidase activity has
been compromised by SW, we would then expect to see an
accumulation of a free oligosaccharide corresponding to
the branched isomer of Man5GlcNAc in the MBCs of SWtreated cells. Results (not shown) demonstrated that 100 µM SW provokes the appearance of only the branched
isomer of Man5GlcNAc in the MBCs of HepG2 cells.
Thus, as expected from its ability to trap large free polymannose type oligosaccharides in the cytosol, 100 µM
SW abolished the appearance of linear Man5GlcNAc in
the MBCs. In summary, results show that incubation of
HepG2 cells with 100 µM SW arrests the cytosolic trimming of free polymannose type oligosaccharides and slows
down their egress from this compartment.
Fig. 4.
Different oligosaccharides appear in the membranebound compartment at different rates. Using the same data that
was used for Fig. 3 the total cellular molar equivalents of the resolved oligosaccharides were displayed as shown at the 1, 2 and 4 h
chase times (top), whereas the percent of each indicated component found to be present in the MBCs is plotted directly below.
The abbreviations are; M9, Man9GlcNAc; M8, Man8GlcNAc; M7,
Man7GlcNAc; M6, Man6GlcNAc; M5, Man5GlcNAc.
[View Larger Version of this Image (59K GIF file)]
Fig. 5.
The effects of
swainsonine on the subcellular trafficking and processing of free polymannose oligosaccharides. HepG2 cells were pulse-radiolabeled and
chased for the indicated
times in the absence (CONTROL) or presence (SW) of
100 µM SW as described in
the legend to Fig. 1. (A) After permeabilization of the
cells free oligosaccharides
were isolated from the cytosolic compartment and quantitated by scintillation counting. After 1 h of chase the
quantity of free oligosaccharides in the cytosol are maximal (see Fig. 1) and this
value has been set at 100%.
During the chase the radioactivity associated with free
oligosaccharides remaining in
the cytosol has been expressed as a percent of the 1 h
chase value. (B) After thin
layer chromatographic resolution of free oligosaccharides
isolated from the cytosolic
compartment (CYTOSOLIC) and membrane-bound compartments (MBC) of cells radiolabeled and chased in the
presence of 100 µM SW,
components (M9, Man9GlcNAc; M8, Man8GlcNAc)
were quantitated as described in the legend to Fig.
3. (C) After permeabilization
of cells that had been chased for 4 h in the presence of 100 µM, SW-free oligosaccharides were isolated from the
cytosolic (CYTOSOL) and
membrane-bound compartments (MBCs) and resolved
by thin layer chromatography on silica-coated plates.
The abbreviations are as defined in the legend to Fig. 1.
[View Larger Version of this Image (24K GIF file)]
), it is possible that this agent inhibits the
normal vesicular traffic of free oligosaccharides through
this compartment and in so doing favors their traffic through the cytosol. To address this question we have performed pulse-chase experiments in the presence of 0.1 µM
SW, a treatment which accurately reproduced the effects
of CCM A on free oligosaccharide trafficking. Accordingly, at this low concentration, we found that SW neither
inhibited the cytosolic processing of free oligosaccharides nor inhibited their loss from the cytosolic compartment. In
addition, as observed with CCM A, 0.1 µM SW provoked
an accumulation of Man5GlcNAc in the MBCs between
the 1- and 8-h chase periods. At 4 h of chase we analyzed
Man5GlcNAc isolated from the MBCs of cells treated with
0.1 µM SW as shown in Fig. 6 (CONTROL), and as can be
seen there is now, in contrast to the situation observed in
the presence of CCM A (Fig. 2), a mixture of the linear
(cytosol-derived) and branched (Golgi-derived) isomers
of Man5GlcNAc. The question now arises as to whether or
not free oligosaccharides modified by Golgi enzymes, observed in the presence of SW, are transported through the
Golgi as free oligosaccharides, or are in fact N-linked to
glycoproteins while in transit through this compartment
before their being released from the peptide backbone in
an upstream degradative compartment. To resolve this
problem we added leupeptin, an inhibitor of lysosomal
protein degradation (Bond and Butler, 1987
) which does
not affect the vesicular import of material into lysosomes
(Rohrer et al., 1995
), to the SW-containing chase medium. As shown in Fig. 6 the quantity of the branched isomer of
Man5GlcNAc is reduced in a dose-dependent manner
without any affect being observed on the recovery of its
linear counterpart. Therefore by using 0.1 µM SW to provoke an accumulation of Man5GlcNAc in the MBCs of
HepG2 cells, we demonstrate that the two isomers of this
free oligosaccharide have different origins. Results indicate that the bulk of free Man5GlcNAc produced by Golgi
enzyme modifications (branched isomer) observed in the
MBCs of SW-treated cells arises from its passage through
the Golgi apparatus while N-linked to glycoprotein and
not as a free oligosaccharide. Thus, although we cannot
rule out the possibility that small amounts of free oligosaccharides are transported from the ER to the Golgi apparatus our results demonstrate that if this pathway does exist
it represents only a minor trafficking route for free oligosaccharides in HepG2 cells.
Fig. 6.
Leupeptin inhibits the formation of the branched isomer of Man5GlcNAc in swainsonine-treated HepG2 cells. HepG2
cells were pulse-radiolabeled and chased for 4 h in the presence
of 0.1 µM SW (CONTROL). Where indicated the chase media
were supplemented with leupeptin (LEU) at either 100 or 300 µg/ml. After permeabilization of the cells and isolation of free
oligosaccharides from the MBCs the resulting radioactive components were resolved by HPAEC as described in Materials and
Methods. The region of the chromatograph corresponding to the
elution times of the linear (LINEAR) and branched (BRANCHED)
isomers of Man5GlcNAc is shown.
[View Larger Version of this Image (29K GIF file)]
),
therefore if autophagy were responsible for the clearance
of free oligosaccharides from the cytosol, we would expect
this reagent to cause an accumulation of fully trimmed
free oligosaccharides in this compartment. Fig. 7 reveals
this not to be the case and in fact after 8 h of chase, 3-MA
had no effect on the clearance of free oligosaccharides
from the cytosolic compartment of HepG2 cells. However,
we did note that 3-MA caused an accumulation of free oligosaccharides in the MBCs, although this accumulation
was not as large as that observed in the presence of CCM
A, it is known that 3-MA interferes with the acidification
of lysosomes (Caro et al., 1988
). Asparagine has been
shown to block the fusion of autophagosomes with the lysosomal compartment (Hoyvik et al., 1991
) and would,
if autophagy were responsible for the clearance of cytosolic oligosaccharides, lead to the accumulation of fully
trimmed free oligosaccharides within MBCs. Again Fig. 7
demonstrates that this was not the case and shows that the
chase incubation performed in the presence of asparagine
is not significantly different from the control chase incubation.
Fig. 7.
Inhibitors of macroautophagy do not affect the clearance of free oligosaccharides from the cytosol of HepG2 cells.
Pulse-chase experiments were performed exactly as described for
Fig. 1. Control or CCM A-treated cells were pulse radiolabeled
and chased for 8 h in the presence of CCM A, 3-methyladenine
(3-MA), or asparagine (Asn). After permeabilization of the cells
as described in Materials and Methods, radioactivity associated
with free oligosaccharides isolated from the cytosolic (CYTOSOL) and membrane-bound compartments (MBCs) was assayed
by scintillation counting.
[View Larger Version of this Image (30K GIF file)]
). Fig. 8 A demonstrates that when HepG2 cells are chased in the presence of deoxyglucose and sodium azide the total molar equivalents of free oligosaccharides within the cytosol remains approximately constant when compared to that observed during control chase periods. Thin layer chromatographic examination of the cytosol- and MBC-derived
oligosaccharides generated in cells treated with inhibitors
of mitochondrial respiration for 8 h is shown in Fig. 8 B
and shows that although cytosolic demannosylation of free oligosaccharides has been completed in drug-treated cells,
the transfer of the cytosolic terminal digest product into an
MBC is severely impaired leading to a marked cytosolic
accumulation of Man5GlcNAc. We observed essentially
identical results when we performed chase incubations in
the presence of 10 µg/ml oligomycin, a specific inhibitor of
the mitochondrial H+/ATPase (Ziegler and Penefsky, 1993
)
(results not shown).
Fig. 8.
The effects of cellular ATP depleting agents on the
clearance of free oligosaccharides from the cytosol of HepG2
cells. HepG2 cells were pulse-radiolabeled and chased for the indicated times as described in the legend to Fig. 1. Where indicated a mixture of deoxyglucose and sodium azide (DOG/AZIDE)
was added to the chase medium 45 min after the onset of the
chase period (to give a final concentration of 10 mM for each
drug). (A) After permeabilization of the cells free oligosaccharides were prepared from the cytosolic fraction and the molar
equivalents of these components were calculated as described in
the legend of Fig. 3. As the quantity of free oligosaccharides
reaches maximal levels after 1 h of chase this value was set at
100% for both the control and drug-treated cells. (B) Free oligosaccharides isolated from the cytosolic compartment (CYTOSOL) and membrane-bound compartments (MBCs) derived
from cells chased for 8 h in the presence and absence of DOG/
AZIDE were separated by thin layer chromatography on silicacoated plates as described in the legend to Fig. 1. The abbreviations are as defined in the legend to Fig. 1. The oligosaccharide
migrating as Man6GlcNAc is known to comprise a mixture of
Glc1Man5GlcNAc and Man6GlcNAc (Moore and Spiro, 1994); the
former is the likely degradation product of Glc1Man9GlcNAc by
the cytosolic mannosidase.
[View Larger Version of this Image (32K GIF file)]
-mannosidase with an acidic pH optimum. Although the lysosome best fits these criteria endosomes also possess a vacuolar H+/ATPase (Mellman et al.,
1986
), and in addition they are known to contain acidic hydrolases (Authier et al., 1994
). To determine the nature of
the MBC in which cytosolic-free oligosaccharides are degraded, we have fractionated HepG2 cell homogenates on
a Percoll gradient known to be able to resolve endosomes
from lysosomes (Rijnboutt et al., 1992
). Initially we chose
to fractionate HepG2 cells after treatment with CCM A,
however results presented in Fig. 9 A demonstrate that
when compared to subcellular fractionation of untreated
cells this reagent caused a marked change in the distribution of the lysosomal marker enzyme,
-hexosaminidase, along the Percoll gradient while not disturbing the distributions of either the Golgi or endoplasmic reticulum
marker enzymes. When CCM A-treated cells were pulse
radiolabeled and then fractionated on a Percoll gradient
we observed that free oligosaccharides (OS-GN2, not
shown) are mainly localized to a region of the gradient
containing endoplasmic reticulum and Golgi marker enzymes (Fig. 9 B). After 4 h of chase in the presence of
CCM A, free oligosaccharides are now distributed throughout the gradient in similar but not identical fashion to that
observed for the lysosomal enzyme
-hexosaminidase.
-hexosaminidase. Although only a small amount of
free oligosaccharides were recovered from subcellular organelles after 1 h of chase, these components were distributed equally between regions of the gradient containing
the lysosomal marker (fraction IV) and the endoplasmic
reticulum/Golgi markers (fraction II). At this early chase
time the gradient fraction II contained free oligosaccharides bearing 9 and 8 residues of mannose whereas those
occurring in the gradient fraction IV comprised oligosaccharides bearing 7-5 residues of mannose (results not shown). After thin layer chromatography of free oligosaccharides recovered from the Percoll density gradients was
performed, the Man5GlcNAc was recovered and quantitated as shown in the lower part of Fig. 11 A. We were unable to detect this oligosaccharide in the subcellular organelles of pulse-radiolabeled cells, but after 1 h of chase,
small amounts of this component could be detected along
the density gradient but were largely found in the lysosomal region of the gradient. After 4 h of chase the distribution of Man5GlcNAc along the Percoll gradient was indistinguishable from that of total free oligosaccharides and
indicated that this component is uniquely localized in the
lysosomal region of the gradient. HPAEC of the
Man5GlcNAc recovered from the lysosomal region of the
Percoll gradients (fraction IV) revealed that after 1 h of chase this fraction contained the linear isomer of
Man5GlcNAc, indicative of its having been generated by
the cytosolic mannosidase. After 4 h of chase both the linear and branched isomers of Man5GlcNAc could be detected in the lysosomal region of the Percoll gradient. Therefore we show that after 4 h of chase in either the
presence of CCM A or 0.1 µM SW free oligosaccharides
are closely associated with a lysosomal marker enzyme after subcellular fractionation on Percoll density gradients.
Fig. 10.
Subcellular fractionation of SW-treated HepG2 cells
on Percoll density gradients. (Top) Cells treated for 4 h with 0.1 µM SW were homogenized and fractionated on Percoll density gradients as described in the legend to Fig. 9. -d-hexosaminidase
(closed circles),
-1, 4-galactosyltransferase (triangles) and NADPH
cytochrome C reductase (open circles) were assayed to identify
the sedimentation positions of lysosomes, the Golgi apparatus
and the ER, respectively. (Bottom) Cells were treated for 1 h
with 0.1 µM SW, pulse labeled with HRP (open symbols), and
then chased for 4 h (solid symbols) as described in Materials and
Methods. HRP-labeled cells were homogenized and a postnuclear supernatant was fractionated on Percoll gradients. Material
from the gradient was recovered in 20 fractions each of which was
assayed for HRP activity.
[View Larger Version of this Image (32K GIF file)]
Fig. 11.
Characterization
of free oligosaccharides isolated from organelles cosedimenting with lysosomal
marker enzymes on Percoll gradients. (A) HepG2 cells
pretreated with 0.1 µM SW
for 1 h, were pulse radiolabeled with d-[2-3H]mannose
and chased for 1 and 4 h in
the presence of SW. After homogenization of the cells
and fractionation of the postnuclear supernatants on Percoll gradients organelles
were recovered from fractions I-IV (see Fig. 10). Free
oligosaccharides (top) were recovered from the organelles of each fraction and
assayed by scintillation
counting. The results have
been expressed as a percentage of the total free oligosaccharides recovered from the
organelles. The quantities of
radioactivity (cpm × 103)
associated with free oligosaccharides isolated from the organelles were 74.7, 27.9, and 197.3, for the pulse, 1 and 4 h chase times, respectively. (Bottom) After thin layer chromatography of the oligosaccharides from each subcellular fraction the oligosaccharide migrating
as Man5GlcNAc was quantitated by scintillation counting and recovery of this component as a percentage of the total Man5GlcNAc recovered from the subcellular fractions I-IV is represented for the pulse and the two chase times. The asterisk indicates that this component was not detected in the pulse incubation. (B) The Man5GlcNAc isolated from fraction IV of the Percoll density gradients as described above was analysed by HPAEC as described in Materials and Methods. The region of the chromatograph corresponding to the
elution times of the linear (LINEAR) and branched (BRANCHED) isomers of Man5GlcNAc is shown.
[View Larger Version of this Image (38K GIF file)]
Discussion
). At present it is unclear whether
this ER-to-cytosol transport of free oligosaccharides is the
sole mechanism responsible for the appearance of free oligosaccharides in the cytosol. Recently, it has been proposed that newly synthesized glycoproteins may be translocated out of the ER and degraded in this compartment (Wiertz et al., 1996
) by the actions of a cytosolic N-glycanase (Kitajima et al., 1995
; Suzuki et al., 1994
) and the proteasome (Ciechanover, 1994
; Driscoll and Goldberg, 1990
).
Clearly such a phenomenon would also give rise to large,
free polymannose-type oligosaccharides in the cytosol. Whatever the origin of cytosolic oligosaccharides in HepG2
cells radioactivity associated with these components reach
maximal levels at ~1 h of chase and declines thereafter
(Fig. 1, A and B). We have tested the hypothesis that cytosolic-free oligosaccharides are translocated into lysosomes to be degraded. To do this we have performed pulse-chase
experiments in the presence of a vacuolar H+/ATPase inhibitor. Concanamycins (Woo et al., 1992
) are antibiotics which are closely related to bafilomycin A (Bowman et al.,
1988
), an agent which has been extensively used to investigate the acidification of intracellular organelles (Yoshimori et al., 1991
; Yilla et al., 1993
; Clague et al., 1994
). In
HepG2 cells the use of bafilomycin has demonstrated that
vacuolar acidification is required for the transfer of fluid
phase markers from endosomes to lysosomes (van Weert
et al., 1995) and concanamycin B has been shown to perturb the trafficking and processing of glycoproteins in late
Golgi compartments. Presumably then, vacuolar ATPase
inhibitors block lysosomal degradation by inhibiting vesicular transport of certain substrates to the lysosome, and
by increasing intralysosomal pH, thereby reducing acidic
hydrolase activity. Accordingly, we reasoned that this
reagent should allow us to examine the fate of cytosolic oligosaccharides without interference by those oligosaccharides generated during glycoprotein catabolism in
the lysosome. We have shown that CCM A is able to substantially inhibit the SW-provoked appearance of the
branched isomer of Man5GlcNAc in the MBC of HepG2
cells suggesting that glycoconjugates that have traversed the Golgi apparatus and which are destined for the lysosome are stabilized in the presence of this agent (data not
shown). Here we show that despite its ability to block lysosomal glycoprotein degradation CCM A provoked an accumulation of free oligosaccharide in an MBC of HepG2
cells. This accumulation coincided precisely with the loss
of free oligosaccharide from the cytosolic compartment. In addition we were able to show that the structures of
the oligosaccharides that accumulated in an MBC of CCM
A-treated cells were characteristic of their having been
generated by the cytosolic mannosidase. These results
clearly demonstrate the transfer of free oligosaccharides from the cytosol into an MBC. We went on to show that
treating HepG2 cells with 100 µM SW blocked the trimming of cytosolic oligosaccharides by the cytosolic
-mannosidase and inhibited their loss from this compartment.
As in the presence of 100 µM, SW oligosaccharides bearing 9 and 8 residues of mannose are stabilized with only a minor quantity of Man7GlcNAc being detected in the cytosolic compartment we conclude that most, if not all, cytosolic-free oligosaccharides must be derived from a preGolgi compartment (SW does not inhibit either the Golgi
mannosidase I (Elbein et al., 1981
), Fig. 5 C, or ER mannosidase I (Weng and Spiro, 1996
). Our results show that Man7-5GlcNAc oligosaccharides can be cleared from the
cytosol although our results strongly suggest that the
smaller the oligosaccharide the more efficient its clearance
from the cytosol. The small amounts of Man7GlcNAc that
are transferred into the MBC are slowly trimmed during
the chase suggesting that CCM A induced neutralization of degradative organelles is not complete (Fig. 1 B). Although lysosomal enzymes are known not to generate linear isomers of polymannose-type oligosaccharides (Michalski et al., 1990
; Al Daher et al., 1991), it is not clear
what isomer of Man5GlcNAc would be generated from
Man7GlcNAc in the CCM A-sensitive MBC, but it is
interesting to note that analysis of the MBC-derived Man5GlcNAc generated during CCM A chases always led
to the detection of small amounts of an oligosaccharide
that lost one mannose residue upon digestion with the
-1,
2 mannosidase (Fig. 2). In conclusion evidence demonstrates that cytosolic oligosaccharides are partially trimmed
in the cytosol and transferred into a membrane bound
compartment. Concerning the relationship between cytosolic trimming and clearance of free oligosaccharides from
the cytosol two observations have to be discussed. First,
Figs. 1 and 4 indicate that there is an apparent "hold up"
of the trimming of cytosolic oligosaccharides at the
Man7GlcNAc stage. Second, inspection of the rate of free oligosaccharide clearance from the cytosol indicates that
this process is somewhat slower than the rate of appearance of these components in the cytosol (which is apparently complete after 1 h of chase; Fig. 3), a fact that would
lead to a steady accumulation of oligosaccharide material
in this compartment. These two observations may be related. We have preliminary data showing that the 5 mM mannose required to be added to the chase incubation media may both slow down the trimming of cytosolic-free oligosaccharides and slow down their clearance from this
compartment (data not shown). Thus under physiological
conditions it may be that cytosolic trimming is more efficient
allowing a more rapid generation of the Man5GlcNAc,
which may be cleared from the cytosol more efficiently than its more highly mannosylated counterparts.
-hexosaminidase. Because we were interested in
the final destination of cytosolic-free oligosaccharides, we
have not directly addressed the question of whether free
oligosaccharides are in fact transported from the cytosol
directly into the lysosome. It is possible that the cytosol-toMBC oligosaccharide translocation machinery is located on a prelysosomal compartment which ultimately fuses
with lysosomes. In this respect our results (Figs. 1-4) with
the vacuolar ATPase inhibitor, CCM A, do not necessarily
imply that the MBC into which free oligosaccharides are
transported contains a pH-sensitive
-mannosidase. It is
possible that free oligosaccharides may be stabilized in
CCM A-treated HepG2 cells because the MBC into which they have been translocated cannot fuse with lysosomes whose
membrane pH gradient has been perturbed. Indeed subcellular fractionation studies performed on CCM A-treated
cells indicate that substantial amounts of free oligosaccharide are found in the endosomal region of the Percoll gradient suggesting that free oligosaccharides may not be
transported directly into lysosomes. However, some of our
observations suggest that free oligosaccharides may be transported directly into lysosomes. First, in the presence
of CCM A, we were able to detect substantial amounts of
free oligosaccharides bearing 3 and 4 residues of mannose
in the vesicular compartment, suggesting that once translocated into this compartment, these components come
into contact with and are slowly acted upon by an
-mannosidase with a low pH optimum. However, as endosomes may contain an acidic
-mannosidase (Authier et al., 1994
),
this result still leaves the possibility that free oligosaccharides are transported into endosomes and are then rapidly
transferred to lysosomes by vesicular fusion. Although we
cannot rule out this possibility, we noted that even when
cells were chased in the presence of 0.1 µM SW for only 1 h,
the majority of Man5GlcNAc recovered from the density
gradient occurred in the lysosomal fraction and not in the
endosomal fraction. The fact that substantial quantities of
the endosomal marker HRP remain associated with endosomes 4 h after an HRP pulse suggests that fluid transfer between endosomes and lysosomes would not be rapid
enough to account for the absence of free oligosaccharides
in the endosomal compartment, if indeed these components
had been translocated from the cytosol into endosomes,
and then onto lysosomes by vesicular transport.
). Here, despite the fact that HepG2 cells are chased
in complete growth medium, a condition known to inhibit
autophagy, we noted that oligosaccharides are cleared
from the cytosol with a half-life of ~3-4 h. Thus, it is apparent that autophagic sequestration cannot account for the transfer of free oligosaccharides into a vesicular compartment of HepG2 cells. In accordance with this we
found that 3-MA, a well known inhibitor of autophagic sequestration (Seglen and Gordon, 1982
), was without effect
on the loss of oligosaccharide material from the cytoplasm.
Furthermore as autophagic sequestration is a nonselective
process, it should theoretically transfer all cytosolic oligosaccharides irrespective of structure into lysosomes. Our results with CCM A (Fig. 4) and 100 µM SW (Fig. 5) suggest that trimming of polymannose oligosaccharides to at
least Man7GlcNAc is required before they are efficiently
transferred into lysosomes, indicating that free oligosaccharides are not being sequestered into the lysosomal degradative compartment by bulk uptake of the cytosol. We
demonstrate that the cytosol-to-lysosome translocation of
free oligosaccharides is strongly impaired if the cells are chased under conditions known to deplete cellular ATP
levels. This result is not surprising as in the presence of
CCM A or 0.1 µM SW the cytosol-to-lysosome transfer of
free oligosaccharides must occur against a substantial concentration gradient. It remains to be determined whether
this transport process is accomplished by a transporter
molecule or by an as yet unidentified mechanism such as a
type of receptor mediated microautophagy. In conclusion our results suggest that free oligosaccharides are sequestred into lysosomes by an energy requiring process that
displays oligosaccharide specificity.
-mannosidosis (see Discussion in
Tulsiani and Touster, 1987
; and conclusion in Daniel et al.,
1992
). Mannosidosis patients have a genetic deficiency in
lysosomal
-mannosidase (Carroll et al., 1972
) which leads
to severe clinical symptoms. At a biochemical level the
lesion is characterized by the presence of large quantities of free oligosaccharides in the tissues and urine from affected individuals. Surprisingly, however, a substantial
proportion of the oligosaccharides isolated from the urine
of mannosidosis patients possess structures not compatable with their having been formed by the incomplete degradation of complex oligosaccharides derived from lysosomal glycoprotein degradation. In fact the structures of
these oligosaccharides were found to be linear in nature
(Nordén et al., 1974
; Strecker et al., 1976
; Daniel et al.,
1992
), the largest of which being identical to the linear
Man5GlcNAc shown in Fig. 2. The trafficking of free polymannose oligosaccharides from ER to cytosol and, after
processing, into the lysosome may now explain the accumulation of the linear oligosaccharides observed in subjects deficient in lysosomal
-mannosidase, however, how these components gain access to the extracellular fluids
still remains to be elucidated. In fact we have calculated
that in CCM A-treated HepG2 cells 15% of all oligosaccharide structures (including N-linked polymannose-,
complex-, and hybrid-type structures, and free oligosaccharides) occur as free polymannose species indicating that this recently outlined free oligosaccharide trafficking
pathway must process large quantities of material in cells
actively engaged in glycoprotein synthesis (data not shown).
This observation is in line with the fact that the urine of
mannosidosis patients may contain up to 250 mg/liter of
small linear oligosaccharides (Strecker et al., 1976
).
). Alternatively free oligosaccharides
maybe delivered to the cytosol for a purpose other than to
be trimmed in order for their ultimate degradation in the
lysosome. Interestingly, the cytosol contains an actin-binding protein, comitin (Weiner et al., 1993
), which has recently been shown to be also a mannose-binding lectin
(Jung et al., 1996
). It was proposed that while the lectin
moiety of this protein could bind to cytosolically disposed
mannose-containing oligosaccharide lipids of the Golgi/
ER its actin-binding domain could tether it to microfilaments (Jung et al., 1996
), thereby forming a bridge between the cytoskeleton and organelles of the secretory
pathway. Could cytosolic-free oligosaccharides compete
for binding sites on comitin thereby modulating this process?
Received for publication 24 May 1996 and in revised form 16 October 1996.
Address all correspondence to S.E.H. Moore, INSERM U410, 16 rue Henri Huchard 75018 Paris, France. Tel.: 33 1 44856134. Fax: 33 1 42288765.We thank Dr. J.R. Green (Ciba-Geigy Ltd.) for the gift of concanamycin A, Dr. J.-C. Michalski for supplying the two standard Man5GlcNAc oligosaccharides, and Dr. C. Rabouille for critical reading of the manuscript. HPAEC was performed by Dr. Thierry Fontaine (Laboratoire des Aspergillus, Institut Pasteur, Paris, Dir. Dr. J.-P. Latgé).
This work was supported by institutional funding from the Institut National de la Santé et de la Recherche Médicale (INSERM) and by grants from the Association Vaincre les Maladies Lysosomales and a European Community Human Mobility and Research Training Fellowship (to S.E.H. Moore).
CCM A, concanamycin A; DOG, deoxyglucose; GlcNAc, N-acetylglucosamine; Man, mannose; MBC, membrane bound compartment; 3-MA, 3-methyladenine; SLO, streptolysin O; SW, swainsonine.