Department of Cell Biology and Anatomy, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205
The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis-Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- 1-propanol (PDMP), shifted the steady-state distribution of IBV M from the Golgi complex to the ER. This effect was rapid and reversible and was also observed for ERGIC-53 but not for Golgi stack proteins. At the concentration of PDMP used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. Pretreatment with upstream inhibitors partially reversed the effects of PDMP, suggesting that ceramide accumulation mediates the PDMP-induced alterations. Indeed, an increase in cellular ceramide was measured in PDMP-treated cells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics.
THE organelles of the classical secretory pathway
must maintain their identity despite a large flux of
lipids and proteins. Two models have emerged to
explain how proteins can be maintained in specific compartments (Machamer, 1993 The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV),1 is a model protein for studying localization to the early secretory pathway. Immunoelectron microscopy showed that IBV M expressed in the
absence of other IBV proteins was found in the tubulo- vesicular structures at the entry or cis face of the Golgi
stack, as well as the first or second cisterna of the Golgi
stack (Machamer et al., 1990 Independent studies demonstrate that the lipid compositions of membranes differ at each stage of the secretory
pathway (Keenan and Morre, 1970 Materials
FCS was from Atlanta Biologicals (Norcross, GA); fumonisin B1 was
from Calbiochem (La Jolla, CA); chromatography plates were from E. Merck (Darmstadt, Germany); endoglycosidase H (endo H) from New
England Biolabs (Beverly, MA); N-hexanoylsphingosine (C6Cer), lipid
standards, and PDMP were from Matreya (Pleasant Gap, PA); [3H]palmitate (50 Ci/mmol) was from DuPont-NEN (Wilmington, DE); Pro-Mix
(>1,000 mCi/mmol [35S]methionine) was from Amersham Intl. (Arlington
Heights, IL); DME, tissue culture-grade trypsin, and penicillin-streptomycin were from GIBCO BRL (Gaithersburg, MD); all other reagents
were from Sigma Chemical Co. (St. Louis, MO).
Antibodies were obtained as follows: monoclonal anti-Bip, Stressgen
(Victoria, BC, Canada); monoclonal anti-giantin and monoclonal anti-ERGIC-53, Hans-Peter Hauri (Basel, Switzerland); polyclonal anti- Methods
Cell Culture.
Tissue culture cells were grown in DME supplemented with
5% (BHK-21) or 10% (Vero) FCS. Cells were grown at 37°C in an atmosphere of 5% CO2.
Pulse-Chase Labeling.
Pulse-chase experiments were performed as
previously described (Rosenwald et al., 1992 Indirect Immunofluorescence Microscopy.
These experiments were performed as previously described (Swift and Machamer, 1991 Lipid Synthesis Assays.
Cells were seeded onto 6-cm plastic dishes 2 d
before the experiment so that they were 90% confluent the day of the experiment. Cells were incubated in DME supplemented with 50 µg/ml cycloheximide, and half of the dishes were also incubated with 5 mM Inhibition of Sphingolipid Synthesis by PDMP but Not
To test whether ongoing sphingolipid synthesis was necessary for the correct localization of the IBV M protein, we
used three inhibitors of sphingolipid synthesis:
To express the IBV M protein, BHK-21 cells were infected with a recombinant vaccinia virus encoding IBV M
(Machamer and Rose, 1987
PDMP Treatment Does Not Mislocalize Golgi Markers,
but Does Mislocalize ERGIC-53
We next asked if PDMP mislocalized other Golgi markers.
Cells were infected and treated as described above and
then stained with antibodies to two integral membrane
proteins of the Golgi stack, giantin (Linstedt and Hauri,
1993
We then looked at a protein which cycles through the
cis-Golgi. As we did not have access to antibodies that
crossreact with known markers in BHK-21 cells, we examined ERGIC-53 in Vero cells. IBV M localizes to the CGN
in these cells and is also redistributed to the ER by PDMP
(data not shown). ERGIC-53 is an IC protein that cycles
between the ER, IC, and Golgi stack (Lippincott-Schwartz et al., 1990
PDMP but Not The above immunofluorescence results suggested that
PDMP might generally alter the distribution of proteins
that have a dynamic localization mechanism. Interestingly,
it has been shown that PDMP slows the rate of both anterograde vesicular traffic (Rosenwald et al., 1992
The PDMP Effect on Anterograde Traffic Is
Ameliorated by Pretreatment with Either Fig. 6 shows that neither Pretreatment with To test whether ceramide was likely to mediate the PDMP-induced mislocalization of IBV M, indirect immunofluorescence was performed as above with cells pretreated with
either
PDMP Increases Cellular Ceramide Levels
The pretreatment experiments suggested that increased
levels of ceramide mediate the PDMP-induced effects on
IBV M localization. Ceramide can act as a second messenger (for review see Hannun, 1994
A Soluble Analogue of Ceramide Does Not Affect the
Localization of IBV M or ERGIC-53
The pretreatment experiments as well as lipid analysis implicated ceramide as the mediator of PDMP effects. If this
were true, addition of a soluble, short-chain analogue of
ceramide might mimic the effects of PDMP. Indeed, one
such cell-permeable analogue of ceramide, N-hexanoylsphingosine (C6Cer), reproduces the effects of PDMP on anterograde traffic of VSV G in CHO cells (Rosenwald and
Pagano, 1993
Steady-State Localization of IBV M Involves Cycling
through the ER
There are two possible mechanisms to maintain the
steady-state localization of proteins to specific sites along
the secretory pathway: (a) retention in the particular compartment, and (b) retrieval from other compartments (Machamer, 1993
Implicit in both models is the idea that IBV M contains
retrieval information. PDMP-induced ER redistribution was
also observed for ERGIC-53, a dilysine-containing IC
marker known to cycle through the ER (Lippincott-Schwartz
et al., 1990 Interestingly, we did not observe redistribution of two
Golgi stack markers (Man II and giantin) to the ER with
PDMP treatment. Golgi membrane proteins were recently
suggested to be highly mobile (Cole et al., 1996b Ceramide Mediates the Effects of PDMP
Interestingly, it has been shown that myriocin, an inhibitor
of sphingolipid synthesis thought to inhibit the same step
as Accumulation of Endogenous Ceramide by PDMP
Treatment and Exogenous C6Cer Do Not Have the
Same Effects
Based on the pretreatment results, we expected that soluble analogues of ceramide would mimic the effects of
PDMP. Rosenwald and Pagano (1993) It is not yet clear how ceramide induces the changes in
anterograde traffic and protein localization. Recent studies have shown that ceramide (Hannun, 1994; Nilsson and Warren, 1994
).
The retention model proposes that proteins are efficiently anchored in the appropriate compartment. The retrieval
model proposes that proteins are continually recycled from
later compartments. The two models are not mutually exclusive; indeed most proteins within the secretory pathway
probably use both mechanisms for localization, albeit to
differing extents. An example of this is the localization of
the ER resident protein BiP, which contains a KDEL retrieval signal but is only slowly secreted when this signal is
removed (Munro and Pelham, 1987
). This suggests that other parts of the molecule may contain retention information.
; Sodeik et al., 1993
). We will
refer to this region as the cis-Golgi network (CGN; Mellman and Simons, 1992
). Defined in this way, we would
consider the CGN to at least partially overlap with the intermediate compartment (IC), defined by such markers as
ERGIC-53 or p58 (Schweizer et al., 1988
; Lahtinen et al.,
1996
). The first transmembrane domain of IBV M is sufficient to target chimeric proteins to the CGN (Swift and
Machamer, 1991
; Machamer et al., 1993
). Although the
transmembrane domains of other Golgi proteins also contain targeting information, no consensus motif for localization has been identified (for review see Colley, 1997
). We
are intrigued by the possibility that the targeting of Golgi membrane proteins may in part depend on interactions
between their transmembrane domains and specific membrane lipids.
; Cluett et al., 1997
; for
review see van Meer, 1993
). Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), the precursor to all gangliosides, are one class of lipids thought to
increase in relative concentration through the secretory
pathway. Ceramide, the precursor of sphingolipids, is synthesized in the ER. In rat liver, ceramide is converted into the different classes of sphingolipids by enzymes localized
to the CGN and the cis- and medial-Golgi cisternae (Futerman et al., 1990
; Futerman and Pagano, 1991
). When
expressed from a recombinant vaccinia virus, IBV M is localized to the CGN in several cell types, and its localization
presumably overlaps with that of SM and GlcCer synthase
activities. This led us to speculate that there may be a link
between sphingolipid synthesis and the localization of
IBV M. To address this question we tested the effects of
three sphingolipid synthesis inhibitors on the steady-state
localization of IBV M. We observed a dramatic redistribution
of IBV M induced by one of these inhibitors, the glucosylceramide analogue d,l-threo-1-phenyl-2-decanoylamino- 3-morpholino-1-propanol (PDMP). Use of upstream inhibitors coupled with lipid analysis suggested that the PDMP
effects are mediated by the accumulation of the precursor
ceramide. Because IBV M can be induced to move to the
ER, we propose that IBV M is at least in part localized by
retrieval mechanisms.
Materials and Methods
-COP,
Jennifer Lippincott-Schwartz (National Institutes of Health, Bethesda,
MD); polyclonal
-mannosidase II, Marilyn Farquhar (University of California at San Diego, La Jolla, CA) and Kelley Moreman (University of
Georgia, Athens, GA); polyclonal antibodies to IBV M and vesicular stomatitis virus (VSV) were prepared as described (Machamer and Rose, 1987
;
Weisz et al., 1993
, respectively); Texas red- and FITC-conjugated secondary antibodies, Jackson ImmunoResearch (West Grove, PA).
; Weisz et al., 1993
). Briefly,
cells were plated in 35-mm dishes the night before the experiment to be
90% confluent the next day. VSV (San Juan strain, Indiana serotype) was
adsorbed for 30 min in 0.5 ml of serum-free DME. At 4 h after infection,
the cells were starved for methionine for 15 min. Cells were then pulsed
for 5 min with 50 µCi 35S-Pro-mix and chased for the indicated amount of
time in the presence or absence of the indicated drugs. The isopropanol
carrier alone had no effect on the rate of transport of VSV G protein. After the chase, cells were washed once with cold PBS and lysed in detergent. VSV G protein was then immunoprecipitated, treated with endo H as described (Rosenwald et al., 1992
), separated by SDS-PAGE, and visualized by fluorography. Endo H-sensitive and -resistant forms of the VSV
G protein were quantitated by densitometry.
). Briefly, cells
were infected for 30 min with a recombinant vaccinia virus encoding IBV
M, and the indicated treatments were begun 4 h after infection. For experiments using exogenous ceramides, the soluble short-chain analogues of
ceramide were added to cells as a complex with 0.34 mg/ml defatted BSA as described (Pagano and Martin, 1988
). After treatment, cells were fixed,
permeabilized, and stained with the appropriate antibody. Images were acquired using a microscope (Axioskop; Zeiss, Inc., Thornwood, NY) equipped
with epifluorescence and a CCD camera (Photometrics Sensys, Tucson,
AZ) using IP Lab software (Signal Analytics Corp., Vienna, VA). All images shown are the raw data collected at 1×1 binning with a gain of 1.
CA. 1 h later, fresh medium containing cycloheximide and [3H]palmitate (100 µCi/
dish) was added, along with either 1% isopropanol (control and
CA)
or 100 µM PDMP and 5 mM
CA if indicated. 1 h later, cells were
trypsinized and washed off the plate in 0.8 ml cold PBS. To normalize lipid
levels, 50 µl were removed for a protein assay by the method of Bradford
(1976)
. Lipids were then extracted by the method of Bligh and Dyer
(1959)
. Labeled samples were doped with 10 µg cold ceramide to allow for
visualization. Samples were run on 10 × 10 cm high performance thin-layer chromatography plates with a mobile phase of chloroform/glacial
acetic acid (9:1; Abe et al., 1992
). Plates were sprayed with water to visualize the ceramide bands that were scraped and counted after the addition of scintillation fluid. Alternatively, plates were dipped in 10% 4,5-diphenyloxazole in chloroform for visualization by fluorography (Henderson
and Tocher, 1992
).
Results
CA or FB1 Causes a Mislocalization of IBV M Protein
to the ER
-chloroalanine (
CA; Medlock and Merrill, 1988
), fumonisin B1
(FB1; Wang et al., 1991
), and PDMP (Vunnam and Radin,
1980
; Inokuchi and Radin, 1987
). The biosynthetic pathway for sphingolipids with the sites of inhibition of these drugs is shown in Fig. 1. Both
CA (5 mM) and FB1 (100 µM) inhibited the incorporation of radiolabeled precursors into sphingolipids in BHK-21 cells by >90% (data not
shown). Consistent with previous results (Rosenwald et al.,
1992
), we found that PDMP (100 µM) inhibited GlcCer
synthesis by ~90% and SM synthesis by ~50% in BHK-21
cells (data not shown).
Fig. 1.
Sphingolipid synthesis pathway. The pathway
for sphingolipid biosynthesis
in mammalian cells and the
steps inhibited by the drugs
used in this study are indicated.
[View Larger Version of this Image (18K GIF file)]
). 4 h after infection the cells
were treated with cycloheximide for 1 h to chase newly
synthesized M protein out of the ER. Sphingolipid synthesis inhibitors were then added and the cells incubated for another 1 h before processing for immunofluorescence
(Fig. 2). In control cells, IBV M exhibited a tight, juxtanuclear staining pattern that colocalized with Golgi markers.
The Golgi localization pattern of IBV M was unchanged
by treatment with either
CA or FB1, suggesting that ongoing sphingolipid synthesis is not required for proper localization of IBV M. In contrast, PDMP-treated cells showed
a marked change in the staining pattern of the IBV M protein after 1 h. The localization of IBV M in the presence of
PDMP changed from Golgi to ER based on the absence of
strong juxtanuclear staining and the presence of nuclear
rim staining and a tubulo-reticular staining pattern that
colocalized with ER markers. Treatment of infected cells
with lower concentrations of PDMP had no effect on IBV M
localization and very little effect on SM synthesis (data not
shown). We next tested the kinetics of PDMP-induced
mislocalization of IBV M. Cells were infected as before
and treated for 1 h with cycloheximide. Then cells were
treated with PDMP for varying lengths of time up to 1 h
(Fig. 3). Redistribution was first observed at 15 min and
became maximal at 60 min. This redistribution was rapidly
reversible, such that 30 min after washing out PDMP, IBV M
had moved back to the Golgi region.
Fig. 2.
PDMP but not CA
or FB1 mislocalize IBV M to
the ER. BHK-21 cells infected
with a recombinant vaccinia
virus encoding IBV M were
treated 4 h after infection
with 50 µg/ml cycloheximide for 1 h. Either 5 mM
CA,
100 µM FB1, or 100 µM
PDMP was then added to the
indicated samples for 1 h, at
which time the cells were
fixed and prepared for indirect immunofluorescence with
antibodies directed against
IBV M and Texas red secondary antibodies. For each
experimental group, the
Nomarski image is shown on
the left and the fluorescence
image on the right. Bar, 10 µm.
[View Larger Version of this Image (69K GIF file)]
Fig. 3.
The redistribution of IBV M induced by PDMP was
rapid and reversible. BHK-21 cells infected with a recombinant
vaccinia virus encoding IBV M were treated 4 h after infection
with 50 µg/ml cycloheximide for 1 h. PDMP (100 µM) was added,
and dishes were fixed at the indicated times and prepared for indirect immunofluorescence. After 1 h in PDMP, the remaining
dishes were washed three times with 1 ml DME with serum and
incubated with cycloheximide-containing medium for the indicated time before being prepared for indirect immunofluorescence. For each experimental series, the Nomarski image is shown
on the left and the fluorescence image on the right. Bar, 10 µm.
[View Larger Version of this Image (36K GIF file)]
) and mannosidase II (Man II; Moremen and Robbins, 1991
; Velasco et al., 1993
). Treatment of cells with
100 µM PDMP for 1 h had no effect on the localization of
either protein (Fig. 4), suggesting that the morphology of the Golgi was not greatly altered. We also examined the
localization of
-COP, a peripheral membrane protein of
the stack and CGN involved in vesicular traffic (Oprins et
al., 1993
). As seen in Fig. 4, the distribution of
-COP appeared unaffected in treated cells.
Fig. 4.
PDMP did not affect localization of endogenous Golgi
markers. BHK-21 cells were incubated for 1 h with cycloheximide
and then incubated for 1 h in medium with 1% isopropanol (control) or 100 µM PDMP. Cells were then fixed and prepared for
indirect immunofluorescence with antibodies to Man II, giantin,
or -COP and Texas red secondary antibodies. For each experimental series, the Nomarski image is shown on the left and the
fluorescence image on the right. Bar, 10 µm.
[View Larger Version of this Image (73K GIF file)]
; Schindler et al., 1993
). Vero cells were treated with cycloheximide for 1 h and then with or without 100 µM
PDMP for an additional hour before being fixed and prepared for immunofluorescence (Fig. 5). Similar to the effects on IBV M, PDMP treatment shifted the steady-state
distribution of ERGIC-53 to the ER. In addition, neither
CA nor FB1 had any effect on the localization of ERGIC-53 (data not shown).
Fig. 5.
PDMP induced the
redistribution of the endogenous IC protein, ERGIC-53,
to the ER. Vero cells were
incubated with cycloheximide for 1 h and then incubated for 1 h in the presence
either 1% isopropanol (control) or 100 µM PDMP. Cells
were then fixed and prepared
for indirect immunofluorescence with antibodies to ERGIC-53.
For each experimental group, the Nomarski image is shown on
the left and the fluorescence image on the right. Bar, 10 µm.
[View Larger Version of this Image (90K GIF file)]
CA or FB1 Slows the Anterograde
Traffic of VSV G
) and endocytosis (Chen et al., 1995
) in CHO cells. We tested whether
PDMP also slowed anterograde traffic in BHK-21 cells. We
used a plasma membrane protein, the well-characterized G protein from VSV. A pulse-chase experiment was performed either in the presence or absence of PDMP,
CA,
or FB1. The rate of accumulation of endo H-resistant VSV G
was used as an assay for the rate of arrival at the medial-Golgi. Neither
CA nor FB1 had any effect on the rate of
anterograde traffic. However, PDMP increased the half-time for transport by ~2.5-fold from 18 to 44 min (Fig. 6).
Fig. 6.
Effect of sphingolipid synthesis inhibitors on transit of
VSV G to the medial-Golgi. BHK-21 cells were infected with
VSV. At 3.25 h after infection, cells were incubated in the presence or absence of 5 mM CA or 100 µM FB1. At 4 h after infection, cells were pulse labeled and chased in the presence or absence of 100 µM PDMP for the indicated times. Cells were lysed
with detergent and VSV G was immunoprecipitated. After treatment of immunoprecipitates with endo H, samples were electrophoresed and autoradiographed. Scanning densitometry was
used to quantitate the amount of endo H-resistant VSV G at
each time point. The data shown are from one representative experiment. PDMP, but not
CA or FB1, slowed arrival of VSV G
at the medial-Golgi by 2.5-fold. Pretreatment with either
CA or
FB1 partially corrected the PDMP effect.
[View Larger Version of this Image (19K GIF file)]
CA or FB1
CA nor FB1 had any effect on
the transit rate of VSV G, suggesting that the decreased
rate with PDMP was not due to a block in the ongoing synthesis of sphingolipids. PDMP inhibits the conversion of
ceramide into glycosphingolipids and sphingomyelin. One
possible explanation for the effects observed with PDMP
was an accumulation of the precursor ceramide.
CA and FB1 act upstream of PDMP (Fig. 1), and the intermediates
in sphingolipid synthesis between these steps and ceramide production are thought to be short lived (Merrill
and Wang, 1986
; Medlock and Merril, 1988). To ask if ceramide accumulation might mediate the PDMP-induced slowing of anterograde traffic, cells were treated with
CA
or FB1 before PDMP. We performed a pulse-chase labeling experiment with cells pretreated with either
CA or
FB1 for 1 h before being chased in the presence or absence
of
CA or FB1 and PDMP (Fig. 6). Pretreatment with the
earlier inhibitors reduced the PDMP-induced slowing of
anterograde traffic by about half, suggesting that accumulation of newly synthesized ceramide at least partially mediates this effect.
CA or FB1 Reduces the
PDMP-induced Mislocalization of IBV M Protein
CA or FB1 for 1 h before the addition of PDMP.
To simplify the quantification, the localization of IBV M
was classified into one of three staining patterns. Class 1 was the most commonly seen pattern in untreated cells,
i.e., tight juxta-nuclear staining that colocalized with Golgi
markers. Class 3 was the most commonly seen pattern in
PDMP-treated cells, i.e., diffuse staining with prominent
nuclear envelope staining that colocalized with ER markers. Class 2 was intermediate between the class 1 and class
3. Whether this pattern represents an overlap of ER and
Golgi staining patterns or an increase in IC staining is not
clear. For these experiments, coded samples were quantified by counting at least 100 cells for each sample. The experiment was performed five times, with one representative experiment shown (Fig. 7). Interestingly, when cells
were quantified in this way, we observed that both
CA
and FB1 caused a slight shift from class 1 to class 2. The
significance of this observation is not clear. PDMP dramatically shifted the distribution of IBV M from mainly
class 1 in the control cells to mainly class 3. Pretreatment
with either
CA or FB1 significantly reduced this shift in
the staining pattern of IBV M, suggesting that accumulation of newly synthesized ceramide mediates the effects of
PDMP on localization of IBV M.
Fig. 7.
Effect of pretreatment with CA and FB1 on PDMP-induced redistribution of IBV M. Cells were prepared for immunofluorescence as described in Fig. 2 with the exception that indicated samples were treated with either 5 mM
CA or 100 µM
FB1 beginning with the cycloheximide chase 4 h after infection.
At 5 h after infection, cells were treated with 1% isopropanol
(control) or 100 µM PDMP. 1 h later the cells were fixed and prepared for indirect immunofluorescence. Random fields from
coded samples were scored for the number of IBV M-expressing
cells that fell into the three classes of staining pattern shown.
Treating cells with FB1 or
CA before PDMP shifted the IBV M
staining pattern towards the control pattern.
[View Larger Version of this Image (63K GIF file)]
) and has been suggested
to be the mediator of some of the effects of PDMP in CHO
cells (Rosenwald and Pagano, 1993
). Furthermore, in several systems, cellular levels of ceramide increased upon treatment with PDMP or one of its more soluble analogues
(for example see Rani et al., 1995
; Posse de Chaves et al.,
1997
). We measured the levels of ceramide after various
treatments by pulse-labeling with [3H]palmitate. After labeling and treatment, cellular lipids were extracted and
run on chromatography plates. The appropriate spots corresponding to ceramide were scraped and counted. Consistent with our hypothesis, Fig. 8 A shows that PDMP
treatment increased the levels of ceramide 2.5-fold over
control levels. Pretreatment with
CA reduced PDMP-
induced ceramide accumulation to roughly half of control
levels, but this level was still threefold higher than cells
treated with
CA alone. Fig. 8 B is a representative fluorograph showing the ceramide region that was quantified
in Fig. 8 A. Interestingly, we noticed the appearance of a
lower migrating ceramide band in PDMP-treated cells.
Fig. 8.
Treatment with PDMP increased the level of newly
synthesized ceramide by 2.5-fold. BHK-21 cells were incubated
with 50 µg/ml cycloheximide and, if indicated, 5 mM CA. After
1 h, media was replaced with medium containing cycloheximide,
100 µCi [3H]palmitate (50 Ci/mmol),
CA if indicated, and either
1% isopropanol (control and
CA) or 100 µM PDMP. After 1 h,
cells were trypsinized to remove them from the dish and the lipids were extracted as described. (A) The spots corresponding to
ceramides on the chromatography plates were scraped, extracted,
and counted. Four experiments were performed, with duplicate
plates for each. Shown are the mean values ± SEM. (B) A representative fluorogram showing the ceramide region of the chromatography plate.
[View Larger Version of this Image (44K GIF file)]
). We first asked whether C6Cer slowed anterograde traffic in BHK-21 cells. A pulse-chase labeling
experiment with VSV-infected cells was performed, with
cells chased in the absence or presence of 25 µM C6Cer.
C6Cer slowed the rate of anterograde traffic in BHK-21
cells ~2.5-fold (data not shown). We went on to use C6Cer
in our indirect immunofluorescence assay. As expected, C6Cer had no effect on the localization of either Man II or
-COP (Fig. 9). However, contrary to our expectation,
C6Cer did not alter the localization of either IBV M or ERGIC-53. This suggests that exogenously added C6Cer and
ceramide generated by treatment with PDMP do not have
the same effects. Two other soluble analogues of ceramide,
C2Cer and C8Cer, were also tested and found to have no
effect on the localization of IBV M (data not shown).
Fig. 9.
Exogenous C6Cer did not redistribute IBV M or ERGIC-53. BHK cells (IBV M, Man II, and -COP) or Vero cells
(ERGIC-53) were infected with a recombinant vaccinia virus expressing IBV M. 4 h after infection the cells were treated with 50 µg/ml cycloheximide. At 5 h after infection cells were incubated
for 1 h in serum-free DME with 0.34 mg/ml defatted BSA with or
without 25 µM C6Cer. Cells were then prepared for indirect immunofluorescence and stained with the indicated antibodies. For
each experimental series, the Nomarski image is shown on the
left and the fluorescence image on the right. Bar, 10 µm.
[View Larger Version of this Image (108K GIF file)]
Discussion
; Nilsson and Warren, 1994
). The redistribution of IBV M induced by PDMP was surprising, as we
expected that if we disrupted its localization, it would
move with "bulk flow" to the plasma membrane. That
IBV M was redistributed to the ER suggests that it has
specific targeting information for retrieval to the ER. Coupled with earlier work in our laboratory on the role of oligomerization of IBV M chimeras in CGN targeting (Weisz
et al., 1993
), our findings suggest that IBV M maintains its
steady-state distribution by both retention and retrieval
(Fig. 10 A). We propose two models to explain the redistribution of IBV M protein induced by PDMP. The simplest model (Fig. 10 B, left arrow) is that the M protein
normally cycles between the ER and the Golgi at a significant rate, as has been shown for ERGIC-53 (Lippincott-Schwartz et al., 1990
; Schindler et al., 1993
). The slowing
of anterograde traffic would cause the protein to shift its
steady-state distribution to the ER, assuming that retrograde traffic is unaffected or perhaps increased by PDMP.
A second possibility is that PDMP disrupts retention of
the M protein, which then cycles back to the ER by normal
retrieval mechanisms (Fig. 10 B, right arrow). These two
models are not mutually exclusive, and both effects of
PDMP may be necessary to redistribute IBV M to the extent observed.
Fig. 10.
Current models
for how IBV M maintains its
steady-state distribution and
how PDMP might alter this
distribution. (A) Evidence presented here shows that
IBV M can be induced to redistribute to the ER, indicating that IBV M has information necessary for traffic to
the ER. We propose that
IBV M normally maintains
its steady-state distribution
by cycling through the ER at
some basal rate. (B) If IBV
M is cycling through the ER
normally, then its localization would depend on the
balance of anterograde and
retrograde traffic rates. As
PDMP slows anterograde
traffic, IBV M could accumulate in the ER when the balance of membrane traffic is disrupted (left arrow). Alternatively, PDMP may act by disrupting the retention of IBV M, causing it to move out of the Golgi (right arrow). It is also possible that redistribution of IBV M requires
both effects of PDMP.
[View Larger Version of this Image (19K GIF file)]
; Schindler et al., 1993
). Double-label immunoelectron microscopy in HeLa cells showed that the distribution of ERGIC-53 and IBV M overlapped significantly, though not completely (Sodeik et al., 1993
). This suggests
that IBV M may in part use the same, as yet unknown,
cellular machinery used by IC proteins to maintain its steady-
state localization. Using deletion mutants and chimeric molecules, dissection of IBV M retention and retrieval information should be possible using PDMP as a tool. The
pathway followed by IBV M during retrieval will also be
important to decipher. Experiments in nontreated cells
suggest that the protein may move through later Golgi
compartments even though its steady-state distribution is
the CGN, because its oligosaccharides are slowly processed (Machamer et al., 1990
).
) and to
cycle through the ER (Cole et al., 1996a
). Our results with
PDMP suggest that cycling of these proteins does not occur during the time scale of our experiments, and/or that
its predominant effect on IBV M is a loss of retention.
CA, reduces the rate of transport of a glycosylphosphatidylinositol-linked protein but not other proteins out
of the ER in yeast (Horvath et al., 1994
). It would be interesting to test whether inhibition of ceramide synthesis in
mammalian cells also slows the rate of transport of glycosylphosphatidylinositol-linked proteins in mammalian cells.
However, neither
CA nor FB1 had an effect on the rate of anterograde traffic of VSV G or the localization of IC,
CGN, or Golgi stack proteins. When we quantified the effects of these two inhibitors on the immunofluorescence
staining pattern of IBV M, we did detect a slight shift toward the intermediate staining pattern (Fig. 7). Whether
this represents an altered distribution pattern or subtle effects on Golgi structure caused by these inhibitors is unclear. From these results we conclude that ongoing sphingolipid synthesis is not required for either normal rates of
anterograde traffic or proper localization of Golgi proteins.
However, the glucosylceramide analogue PDMP, at a concentration that inhibits both GlcCer and SM synthases,
causes a redistribution of IBV M to the ER and a slowing
of anterograde traffic in BHK-21 cells. Why does PDMP
have these effects while
CA and FB1 do not? It is likely
that PDMP exerts its effects by causing the accumulation of ceramide. Other groups have found that the various effects of PDMP and its active analogues were concomitant
with (Uemura et al., 1990
; Shayman et al.; 1991; Rani et al.,
1995
) or actually caused by increases in ceramide concentrations (Abe et al., 1996
; Posse de Chaves et al., 1997
). In
BHK-21 cells treated with 100 µM PDMP for 1 h we measured an increase in newly synthesized ceramide (Fig. 8 A),
showing at least a correlation between levels of newly synthesized ceramide and the effects of PDMP on protein traffic and localization. The fact that pretreatment with either
CA or FB1, both of which block the synthesis of ceramide, can ameliorate the effects of PDMP demonstrates
that ceramide is at least in part the mediator of these effects. However, the effects of PDMP are not completely
abolished by pretreatment, even though
CA pretreatment reduced the level of newly synthesized ceramide by 50% compared to control. One explanation is that ceramide accumulation is only one of the ways that PDMP
exerts its effects. Another possibility is that ceramide is being generated by the degradation of preexisting sphingolipids, e.g., at the cell surface or in lysosomes. This ceramide cannot be made back into sphingolipids because the
enzymes SM and GlcCer synthases are inhibited in the presence of PDMP. Interestingly, the fluorograph of the ceramide bands generated after the various treatments (Fig. 8
B) shows different relative amounts of ceramide species.
We suspect that these ceramides differ in acyl chain lengths
(Abe et al., 1992
) and that this may be an indication of different sources of ceramide, i.e., de novo synthesis or sphingolipid degradation. We are currently investigating this possibility.
showed that like
100 µM PDMP, 25 µM C6Cer decreased the rate of transit
of an itinerant protein to the medial-Golgi in CHO cells.
Consistent with these results, 25 µM C6Cer also slowed anterograde traffic in BHK-21 cells. However, 25 µM C6Cer had no effect on the localization of either the M protein or
ERGIC-53. Other short-chain ceramide analogues (C2-
and C8-ceramide) also had no effect on IBV M localization. How does this fit with our model that ceramide mediates PDMP effects? One explanation is that the effect on
protein localization is independent of the effect on anterograde traffic rates. In this case, it may be that C6Cer and
endogenous ceramide have similar ability to bind and affect proteins that regulate anterograde traffic, but C6Cer is unable to bind or affect proteins that regulate protein localization. It has been shown that some short chain analogues of ceramide do not have the same effects as endogenous ceramide (e.g., Wolff et al., 1994
). A second possibility
is that effects of ceramide are limited to the bilayers in
which its concentration increases, and the difference seen
between endogenous ceramide and exogenous C6Cer is
due to the different intracellular distribution of these molecules. C6Cer would be unlikely to accumulate in compartments where IBV M and ERGIC 53 reside, because it is
rapidly converted to sphingolipids there (Rosenwald et al.,
1992
). Effects on membrane traffic might be more dramatic
in compartments where C6Cer accumulates, possibly the
trans-Golgi and TGN (Pagano et al., 1989
). Consistent with this idea, the rate of movement of VSV G through the
late Golgi (measured by sialylation) was slowed to a much
greater extent by C6Cer than by PDMP (Rosenwald and
Pagano, 1993
).
) and its metabolites (Hakomori, 1990
) play a major role as second
messengers. Preliminary experiments with both the ceramidase inhibitor N-oleoylethanolamine and exogenously added sphingosine suggest that the ceramide breakdown
product sphingosine does not play a role in the localization
of IBV M (Maceyka, M., and C. Machamer, unpublished
observations). Ceramide activates a cytosolic protein phosphatase (Dobrowsky and Hannun, 1992
; Wolff et al., 1994
)
that can be inhibited by okadaic acid. Preliminary experiments suggest that okadaic acid does not block the PDMP-induced changes (Maceyka, M., and C. Machamer, unpublished observations). Ceramide could be activating a protein
kinase (Mathias et al., 1991
), but this kinase has not been
fully characterized. Another possibility is that increased
ceramide within the lipid bilayer directly affects the localization of IBV M. Earlier work from our laboratory showed
that the first transmembrane domain of IBV M can target
chimeras to the CGN (Swift and Machamer, 1991
; Machamer
et al., 1993
). It is possible that CGN bilayers have distinct
lipid domains, and that these domains contain different sets
of proteins, analogous to glycosphingolipid rafts. Segregation of membrane proteins involved in vesicular traffic,
such as SNAREs, would lead to mobile and immobile domains. We hypothesize that under normal conditions, IBV
M would be targeted to an immobile domain and escaped
molecules would be cycled back through the ER. Elevated
levels of ceramide could disrupt these immobile lipid domains, inducing IBV M to cycle. Alternatively, perhaps
ceramide binds to IBV M transmembrane domains, preventing an interaction with immobile lipid domains.
Received for publication 29 August 1997 and in revised form 6 October 1997.
Address all correspondence to Carolyn E. Machamer, Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: (410) 955-1809. Fax: (410) 955-4129. E-mail: carolyn_machamer{at}qmail.bs.jhu.eduWe thank Drs. E. Cluett, D. Raben, and A. Hubbard for useful discussions and the members of the Machamer lab for critical reading of the manuscript. We also thank H.-P. Hauri, M. Farquhar, K. Moremen, and J. Lippincott-Schwartz for antibodies.
This work was supported by grant GM42522 from the National Institutes of Health.
CGN, cis-Golgi network; GlcCer, glucosylceramide; IBV, infectious bronchitis virus; IC, intermediate compartment; PDMP, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; SM, sphingomyelin; VSV, vesicular stomatitis virus.
1. | Abe, A., D. Wu, J.A. Shayman, and N.S. Radin. 1992. Metabolic effects of short-chain ceramide and glucosylceramide on sphingolipids and protein kinase C. Eur. J. Biochem. 210: 765-773 [Abstract]. |
2. | Abe, A., N.S. Radin, and J.A. Shayman. 1996. Inhibition of glucosylceramide synthase by synthase inhibitors and ceramide. Biochim. Biophys. Acta. 1299: 333-341 |
3. | Bradford, M.M.. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 |
4. | Bligh, E.G., and W.J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917 . |
5. | Bretscher, M.S., and S. Munro. 1993. Cholesterol and the Golgi apparatus. Science. 261: 1280-1281 |
6. |
Chen, C.-S.,
A.G. Rosenwald, and
R.E. Pagano.
1995.
Ceramide as a modulator
of endocytosis.
J. Biol. Chem.
270:
13291-13297
|
7. |
Cluett, E.B.,
E. Kuismanen, and
C.E. Machamer.
1997.
Heterogeneous distribution of an unusual phospholipid in the Golgi complex.
Mol. Biol. Cell.
8:
2233-2240
|
8. | Cole, N.B., N. Sciaky, A. Marotta, J. Song, and J. Lippincott-Schwartz. 1996a. Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol. Biol. Cell. 7: 631-650 [Abstract]. |
9. | Cole, N.B., C.L. Smith, N. Sciaky, M. Terasaki, M. Edidin, and J. Lippincott-Schwartz. 1996b. Diffusional mobility of Golgi proteins in membranes of living cells. Science. 273: 797-801 [Abstract]. |
10. | Colley, K.J.. 1997. Golgi localization of glycosyltransferases: more questions than answers. Glycobiology. 7: 1-13 [Abstract]. |
11. |
Dobrowsky, R.T., and
Y. Hannun.
1992.
Ceramide stimulates a cytosolic protein phosphatase.
J. Biol. Chem.
267:
5048-5051
|
12. | Futerman, A.H., and R.E. Pagano. 1991. Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver. Biochem. J. 280: 295-302 |
13. |
Futerman, A.H.,
B. Stieger,
A.L. Hubbard, and
R.E. Pagano.
1990.
Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus.
J. Biol. Chem.
265:
8650-8675
|
14. |
Hakomori, S.-I..
1990.
Bifunctional role of glycosphingolipids. Modulators for
transmembrane signaling and mediators for cellular interactions.
J. Biol.
Chem.
265:
18713-18716
|
15. |
Hannun, Y.A..
1994.
The sphingomyelin cycle and the second messenger function of ceramide.
J. Biol. Chem.
269:
3125-3128
|
16. | Henderson, R.J., and D.R. Tocher. 1992. Thin-layer chromotography. In Lipid Analysis: A Practical Approach. R.J. Hamilton and S. Hamilton, editors. Oxford University Press, Oxford. 65-111. |
17. | Horvath, A., C. Sutterlin, U. Manning-Krieg, N.R. Movva, and H. Riezman. 1994. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. EMBO (Eur. Mol. Biol. Organ.) J. 13: 3687-3695 [Abstract]. |
18. | Inokuchi, J.-I., and N.S. Radin. 1987. Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase. J. Lipid Res. 28: 565-571 [Abstract]. |
19. | Keenan, T.W., and D.J. Morre. 1970. Phospholipid class and fatty acid composition of Golgi apparatus isolated from rat liver and comparison with other cell fractions. Biochemistry. 9: 19-25 |
20. |
Lahtinen, U.,
U. Hellman,
C. Wernstedt,
J. Saraste, and
R.F. Pettersson.
1996.
Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate
compartment protein.
J. Biol. Chem.
271:
4031-4037
|
21. | Linstedt, A.D., and H.-P. Hauri. 1993. Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350kDa. Mol. Biol. Cell. 4: 679-693 [Abstract]. |
22. | Lippincott-Schwartz, J., J.G. Donaldson, A. Schweizer, E.G. Berger, H.-P. Hauri, L.C. Yuan, and R.D. Klausner. 1990. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 60: 821-836 |
23. | Machamer, C.E.. 1993. Golgi retention signals: do membranes hold the key? Curr. Opin. Cell Biol. 5: 606-612 |
24. | Machamer, C.E., and J.K. Rose. 1987. A specific membrane-spanning domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region. J. Cell Biol. 105: 1205-1214 [Abstract]. |
25. | Machamer, C.E., S.A. Mentone, J.K. Rose, and M.G. Farquhar. 1990. The avian coronavirus E1 protein is targeted to the cis Golgi. Proc. Natl. Acad. Sci. USA. 87: 6944-6948 [Abstract]. |
26. |
Machamer, C.E.,
M.M. Grim,
A. Esquela,
S.W. Chung,
M. Rolls,
K. Ryan, and
A.M. Swift.
1993.
Retention of a cis Golgi protein requires polar residues on
one face of a predicted ![]() |
27. |
Mathias, S.,
K.A. Dressler, and
R.N. Kolesnick.
1991.
Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor ![]() |
28. |
Medlock, K.A., and
A.H. Merrill.
1988.
Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster
ovary cells by ![]() |
29. | Mellman, I., and K. Simons. 1992. The Golgi complex: in vitro veritas? Cell. 68: 829-840 |
30. |
Merrill, A.H., and
E. Wang.
1986.
Biosynthesis of long-chain (sphingoid) bases
from serine by LM cells. Evidence for introduction of the 4-trans-double
bond after de novo biosynthesis of N-acylsphinganines.
J. Biol. Chem.
261:
3764-3769
|
31. |
Moremen, K.W., and
P.W. Robbins.
1991.
Isolation, characterization and expression of cDNAs encoding murine ![]() |
32. | Munro, S., and H.R.B. Pelham. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 48: 899-907 |
33. | Nilsson, T., and G. Warren. 1994. Retention and retrieval in the endoplasmic reticulum and Golgi apparatus. Curr. Opin. Cell Biol. 6: 517-521 |
34. |
Oprins, A.,
R. Duden,
T.E. Kreis,
H.J. Geuze, and
J.W. Slot.
1993.
![]() |
35. | Pagano, R.E., and O.C. Martin. 1988. Use of fluorescent analogs of ceramide to study the Golgi apparatus of animal cells. In Cell Biology: A Laboratory Handbook. J.E. Celis, editor. Academic Press, Inc., San Diego. 387-393. |
36. | Pagano, R.E., M.A. Sepanski, and O.C. Martin. 1989. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J. Cell Biol. 109: 2067-2079 [Abstract]. |
37. |
Posse de Chaves, E.I.,
M. Bussiere,
D.E. Vance,
R.B. Campenot, and
J.E. Vance.
1997.
Elevation of ceramide within distal neurites inhibits neurite
growth in cultured rat sympathetic neurons.
J. Biol. Chem.
272:
3028-3035
|
38. |
Rani, C.S.S.,
A. Abe,
Y. Chang,
N. Rosenzweig,
A.R. Saltiel,
N.S. Radin, and
J.A. Shayman.
1995.
Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-independent kinases.
J. Biol. Chem.
270:
2859-2867
|
39. |
Rosenwald, A.G., and
R.E. Pagano.
1993.
Inhibition of glycoprotein traffic
through the secretory pathway by ceramide.
J. Biol. Chem.
268:
4577-4579
|
40. | Rosenwald, A.G., C.E. Machamer, and R.E. Pagano. 1992. Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway. Biochemistry. 31: 3581-3590 |
41. | Schindler, R., C. Itin, M. Zerial, F. Lottspeich, and H.-P. Hauri. 1993. ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, carries an ER retention motif. Eur. J. Cell Biol. 61: 1-9 |
42. | Schweizer, A., J.A.M. Fransen, T. Baechi, L. Ginsel, and H.-P. Hauri. 1988. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulovesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol. 107: 1643-1653 [Abstract]. |
43. |
Shayman, J.A.,
G.D. Deshmukh,
S. Mahdiyoun,
T.P. Thomas,
D. Wu,
F.S. Barcelon, and
N.S. Radin.
1991.
Modulation of renal epithelial cell growth by
glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.
J. Biol. Chem.
266:
22968-22974
|
44. | Sodeik, B., R.W. Doms, M. Ericsson, G. Hiller, C.E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths. 1993. Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks. J. Cell Biol. 121: 521-541 [Abstract]. |
45. | Swift, A.M., and C.E. Machamer. 1991. A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein. J. Cell Biol. 115: 19-30 [Abstract]. |
46. | Uemura, K.-I., E. Sugiyama, C. Tamai, A. Hara, T. Taketomi, and N.S. Radin. 1990. Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts. J. Biochem. (Tokyo). 108: 525-530 [Abstract]. |
47. | van Meer, G.. 1993. Transport and sorting of membrane lipids. Curr. Opin. Cell Biol. 5: 661-673 |
48. |
Velasco, A.,
L. Hendricks,
K.W. Moremen,
D.R.P. Tulsiani,
O. Touster, and
M.G. Farquhar.
1993.
Cell type-dependent variations in the subcellular distribution of ![]() |
49. | Vunnam, R.R., and N.S. Radin. 1980. Analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain. Chem. Phys. Lipids. 26: 265-278 |
50. |
Wang, E.,
W.P. Norred,
C.W. Bacon,
R.T. Riley, and
A.H. Merril Jr..
1991.
Inhibition of sphingolipid biosynthesis by fumonisins: implications for diseases
associated with Fusarium moniliforme.
J. Biol. Chem.
266:
14486-14490
|
51. | Weisz, O.A., A.M Swift, and C.E. Machamer. 1993. Oligomerization of a membrane protein correlates with its retention in the Golgi complex. J. Cell Biol. 122: 1185-1196 [Abstract]. |
52. |
Wolff, R.A.,
R.T. Dobrowsky,
A. Bielawska,
L.M. Obeid, and
Y. Hannun.
1994.
Role of ceramide-activated protein phosphatase in ceramide-mediated
signal transduction.
J. Biol. Chem.
269:
19605-19609
|