From the
The mouse hepatitis virus M protein is a triple spanning
membrane glycoprotein that, when expressed independently, localizes to
trans-Golgi as well as to the trans-Golgi network
(TGN). Passage of this protein from the endoplasmic reticulum through
the intermediate compartment to the late Golgi and TGN can be
conveniently followed by analyzing its O-linked sugars.
Using pulse-chase analyses we studied the oligomerization of the M
protein in sucrose gradients. The Golgi and TGN forms migrated as large
heterogeneous complexes, whereas the endoplasmic reticulum and
intermediate compartment forms of the protein appeared to migrate as
monomer. Moreover, a mutant of the M protein lacking the 22
COOH-terminal amino acids, that is transported to the plasma membrane,
gave rise to similar complexes, albeit smaller in size, that persisted
at the plasma membrane.
We propose that the trans-Golgi/TGN
retention of the MHV-M protein is governed by two mechanisms:
oligomerization possibly mediated by the transmembrane domains and
binding of its cytoplasmic tail to cellular factors in trans Golgi/TGN.
Proteins that are retained at intracellular locations may
contain signals present in their primary sequence that specify their
retention. Two well documented examples of such a signal are the KDEL
sequence (ER;
No such signal has yet been identified for Golgi
proteins. Mutational analyses have suggested that the transmembrane
domain of many Golgi proteins contains sufficient information for
retention (see e.g. Munro, 1991; Nilsson et al.,
1991; Swift and Machamer, 1991; Burke et al., 1992; Colley
et al., 1992; Teasdale et al., 1992; Wong et
al., 1992). However, these membrane-spanning domains appear not to
share sequence homology.
One way to explain retention by means of
transmembrane regions is by ``kin recognition'' (Nilsson
et al., 1993, 1994). According to this model, upon their
arrival at the appropriate cisterna of the stack, Golgi proteins will
form large heterooligomers possibly mediated by their membrane-spanning
domains. Within any one cisterna these oligomers would comprise the
different proteins that localize to this particular cisterna. Nilsson
et al. (1993) have argued that the retention may also be
facilitated by the cytoplasmic tails of these proteins possibly by
binding to a cytosolic intercisternal matrix. A consequence of this
retention mechanism is that the ensuing complexes are thought to be so
large that they are physically excluded from transport vesicles
(Nilsson et al., 1993, 1994). Although not mutually exclusive,
the putative formation of oligomers in the Golgi complex may also be
induced by the microenvironment of a specific Golgi cisterna, for
instance its lipid composition (Machamer, 1993). So far, only one
example of complex formation or detergent insolubility of a
Golgi-retained protein has been described in vivo (Weisz
et al., 1993).
Other studies have emphasized that Golgi
membrane proteins have generally shorter and less hydrophobic
transmembrane domains compared with plasma membrane proteins and that
this may explain retention of the former (Masibay et al.,
1993; Bretscher and Munro, 1993). Bretscher and Munro (1993) pointed
out that the plasma membrane is relatively enriched in cholesterol, a
lipid that tends to make the bilayer ``thicker.''
Golgi-resident proteins, according to this view, will be retained
simply because they are sorted away from the thick bilayers. In three
cases experimental evidence appeared to be consistent with this
hypothesis (Munro, 1991; Swift and Machamer, 1991; Masibay et
al., 1993).
The M protein of coronaviruses has been extensively
used as a tool to study Golgi retention (see above; Swift and Machamer,
1991; Armstrong et al., 1990; Armstrong and Patel, 1991). The
overall structure is well conserved among different coronaviruses; it
invariably contains a short luminal domain of about 25 amino acids that
is either O (mouse hepatitis virus, MHV)- or
N-glycosylated (IBV-M), three membrane-spanning domains and a
long amphiphilic cytoplasmic domain (see e.g. Rottier et
al., 1986). The O-glycosylation of the MHV-M protein has
been analyzed extensively (Niemann et al., 1984; Tooze et
al., 1988). In SDS-PAGE one can distinguish up to five
electrophoretically different forms, which we have referred to as
M
The VV recombinant expressing the IBV-M protein (vvIBV-M) was a kind
gift of Carolyn Machamer (John Hopkins University, Baltimore) and has
been described before (Machamer et al., 1990). The following
antibodies were used to detect these proteins: the polyclonal anti-MHV
serum (Rottier et al., 1981) and a peptide serum recognizing
the 18 COOH-terminal amino acids of the MHV-M protein (Krijnse Locker
et al., 1992b); a rabbit serum raised against the VSV-G
protein was a kind gift of Jean Gruenberg (University of Geneva,
Switzerland); the rabbit peptide serum recognizing the IBV-M protein
(Machamer et al., 1990) was kindly provided by Carolyn
Machamer. To detect sialyltransferase in the SA:48 cells we used the
monoclonal antibody P5D4 recognizing the VSV-G P5D4 epitope (Kreis,
1986).
Except for the IBV-M protein (see below), infected cells were
pulse-labeled at 5.30 h post-infection with 100-200 µCi of
COS-1 cells were infected with vvMHV-M expressing the M protein. The
cells were pulse-labeled and chased and lysed with the same detergent
combination used previously to demonstrate the M/S complexes,
consisting of 0.5% Nonidet P-40 and 0.5% sodium deoxycholic acid
(Opstelten et al., 1994a; see also ``Materials and
Methods''). The lysates were loaded onto 15-30% sucrose
gradients, centrifuged, and after fractionation the M protein was
immunoprecipitated from each fraction. The unglycosylated ER form of
the M protein (M
We next quantitated the distribution of the
immunoprecipitated forms of the M protein over the different fractions
by PhosphorImager (see ``Materials and Methods''). As
expected from Fig. 3, after the pulse labeling the bulk of the
label was present at the top of the gradient, whereas after the chase
the M protein was found more or less evenly distributed over the
gradient with a slight increase toward the bottom (Fig. 4). In
the bottom fraction we consistently noted a 25-kDa protein (as well as
two other proteins that were not co-migrating with the M protein),
probably derived from the vaccinia virus infection, that almost
co-migrated with the M
This tail-less mutant
protein (called M
The M protein was pulse-labeled for 15 min and chased for 1 h both
in the presence of BFA. Consistent with our earlier data (Krijnse
Locker et al., 1992a, 1992b) the O-glycosylation was
much faster in the presence of the drug and the M protein was already
mainly converted to the M
Another
striking difference between the M protein and molecules such as VSV-G
was that complex-formation did not result in dimers or trimers, but
rather in heterogeneous and large oligomers. We estimate that the
largest complexes are roughly 1000 kDa. this would argue that they may
contain about 40 molecules of the M protein and are therefore
considerably larger than the trimers of the VSV-G and the influenza HA
protein. At present it is not known whether the heterogeneity we
observed is due to the fact that these complexes are unstable under our
experimental procedures or whether it is an inherent characteristic of
the M complexes. The sizes of the aggregates we describe were not
appreciably different after cross-linking, nor were they affected by pH
(not shown). Also, when we tested different detergents, the M protein
migrated as a monomer in the presence of all (that were tested like
Nonidet P-40, Triton X-100, sodium deoxycholic acid, and octyl
glucoside) except one. The exception was CHAPS in which the M protein
behaved identical as in our sodium deoxycholic acid/Nonidet P-40
mixture.
The formation of the complexes was neither affected by
nocodazole nor by cytochalasin D, suggesting that the cytoskeletal
elements sensitive to these drugs are not important for this process
(not shown).
Recent data from our laboratory have shown that when the
M and S protein are co-expressed, the formation of multimers (now
containing both M and S) may start as early as the IC ( i.e. pre-Golgi). Subsequently, these (preformed) M/S complexes appear
to be transported in a multimeric form to the Golgi and possibly to the
TGN, where they are retained. These data imply, as for the M protein
alone, that the complex formation is not the sole determinant of their
retention.
The medial Golgi enzymes NAGT I and Man II can functionally
interact in vivo. Although their intracellular distribution
partially overlaps with GalTf, they appear not interact with this
latter enzyme (Nilsson et al., 1994). The coronavirus M
protein may have no cellular template to interact with. If one assumes
that our complexes are predominantly due to protein-protein
interactions (see above), it seems most likely that the MHV-M oligomers
we detect are composed of the M protein only. The simplest
interpretation of our available data is that the intracellular
retention of this protein is dependent on both this
``self-association'' and on the interaction of the
cytoplasmic tail with components on the cytosolic side of the TGN,
consistent with the kin recognition model (Nilsson et al.,
1993).
We thank Kai Simons, Tommy Nilsson, Klaus Fiedler,
Sanjay Pimplikar, and Gareth Griffiths for many helpful discussions and
critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
Pelham, 1988; Tang et
al., 1992) and the signal consisting of two positively charged
amino acids located in the cytoplasmic tails of both type I and II
resident ER proteins (Jackson et al., 1990; Schutze et
al., 1994).
through M
. Since these forms correspond to
the arrival of the protein in different intracellular compartments (see
Fig. 1
; Krijnse Locker et al., 1992a, 1994a), the extent
of O-glycosylation can be used as a marker of transport of the
M protein. When expressed independently the IBV-M protein localizes to
the cis-Golgi region, where it co-localizes with markers of
the IC (Machamer et al., 1990; Sodeik et al., 1993;
Klumperman et al., 1994). In contrast, and for reasons that
remain elusive, the MHV-M protein localizes to the trans side of the
Golgi and the TGN (Krijnse Locker et al., 1992a; Klumperman
et al., 1994). This difference in localization also seems to
have implications for their retention signals. In the IBV-M protein,
but not in MHV-M, the first transmembrane domain appears to be
sufficient for retention (Swift and Machamer, 1991), whereas the
cytoplasmic tail of the MHV-M protein, in contrast to that of IBV-M,
appears to contain information for retention (Armstrong and Patel,
1991; Krijnse Locker et al., 1994b). In this study we have
analyzed the oligomerization of the MHV-M protein with the aim of
testing the hypothesis that retention of this protein may correlate
with the formation of detergent-insoluble aggregates.
Figure 1:
Representation of the different
O-glycosylated forms of the M protein as detected by SDS-PAGE,
their sugar modification, and the different compartments in which the
modifications have been shown to occur. The Mform has been
placed between parentheses, because this form does not
accumulate to detectable amounts in
vivo.
Cells, Viruses, and Antibodies
COS-1 cells were
maintained in Dulbecco's modified Eagle's medium with 5%
fetal calf serum. SA:48 cells, a kind gift of Tommy Nilsson (Imperial
Cancer Research Foundation, London), were kept in Dulbecco's
modified Eagle's medium, 10% FCS containing 200-400
µg/ml Geneticin (Life Technologies, Inc., Paisley, Scotland). The
vaccinia virus (VV) recombinants expressing the wild-type MHV-M protein
(vvMHV-M) and the M protein lacking the cytoplasmic tail
(vvMHV-MCOOH) have been described (Krijnse Locker et al.,
1992b; Krijnse Locker et al., 1994b). The VSV-G protein was
expressed using a VV recombinant expressing the tsO45 mutant of the G
protein under the control of an early, 7.5 kDa, VV promotor
(vvVSV-GtsO45; a kind gift of Robert Doms, University of Pennsylvania).
Metabolic Labeling and Velocity Gradient
Centrifugation
The M and G proteins were expressed by infecting
COS-1 cells grown in 35- or 60-mm dishes with the respective VV
recombinants as described (Krijnse Locker et al., 1992b).
S Express-label (NEN Dupont GmbH, Dreiech, Germany) for
the indicated time. The cells were either lysed immediately or chased
for 2 h before lysis. The lysis buffer consisted of 50 m
M Tris-Cl, pH 8.0, 62.5 m
M EDTA, 0.5% Nonidet P-40
(Sigma-Aldrich, Deisenhofen, Germany), 0.5% sodium deoxycholic acid
(Sigma). After lysis on ice, the samples were layered on top of a
continuous 15-30% (w/v) sucrose gradient layered on top of a
cushion of 0.2 ml of 60% (w/v) sucrose. The sucrose solutions were in
50 m
M Tris-Cl, pH 8.0, 62.5 m
M EDTA, 0.1% of Nonidet
P-40, and 0.1% sodium deoxycholic acid. The gradients were spun for
15-16 h at 40,000 rpm and 4 °C in a SW 60 rotor. Each
gradient was fractionated in ten fractions of 450 µl, each fraction
was diluted with 500 µl of lysis buffer and the labeled proteins
they contained were immunoprecipitated with 1-3 µl of the
respective antibody. The immunoprecipitation was carried out as
described (Krijnse Locker et al., 1992b), and the proteins
were analyzed in a 10 or 15% SDS-PAGE. The treatment with brefeldin A
was carried out as described (Krijnse Locker et al., 1992a).
The endo-
- N-acetylglucosaminidase H treatment of the
VSV-G protein was as described earlier (Klumperman et al.,
1994).
Western Blotting and Enhanced Chemiluminescence
(ECL)
Since the IBV-M protein was difficult to detect by
immunoprecipitation, this protein was analyzed by ECL. COS cells
infected with vvIBV-M were lysed as above at 9 h post-infection. The
lysate was layered on a sucrose gradient and spun as described above,
but the M protein present in the collected fractions was
trichloroacetic acid-precipitated as follows; each fraction of the
gradient was adjusted to 10% sucrose with lysis buffer, and an equal
volume of 20% trichloroacetic acid, 50% acetone, and 10 µg of
myoglobin as carrier was added. The samples were left on ice for 60
min, spun for 15 min at 14,000 rpm at 4 °C, and the pellets were
dissolved in Leammli sample buffer. The precipitates were run in a 15%
SDS-PAGE and transferred to nitrocellulose (ECL, Hybond, Amersham
International, Buckinghamshire, United Kingdom). The blot was
extensively blocked with 5% milk powder in phosphate-buffered saline
containing 0.1% Tween 20. The blot was incubated for 1 h with the
anti-IBV-M peptide serum at a 1/1000 dilution. The secondary antibody,
goat anti-rabbit conjugated to horseradish peroxidase was from Cappel
(Cooper Biomedical, West Chester, PA). The IBV-M protein was detected
by ECL (Amersham) according to the instructions of the manufacturer.
Estimation of the Size of the Complexes
For the
estimation of the size of the M protein complexes two proteins with
known swere used; catalase
(11.3 s
) and thyroglobulin
(19.3 s
) were centrifuged
as described above, the proteins in the gradient fractions were
trichloroacetic acid-precipitated, run in SDS-PAGE, and stained with
Coomassie Blue. The size estimation of the M protein and the mutant
protein M
COOH complexes was carried out according to Martin and
Ames (1961).
Surface Immunoprecipitation
The surface
immunoprecipitation of the mutant M protein MCOOH was done as
described (Krijnse Locker et al., 1994b).
Electron Microscopy
SA:48 cells were infected with
the recombinant VV expressing the MHV-M protein as described (Krijnse
Locker et al., 1992b). At 6 h post-infection 16 nm bovine
serum albumin-gold at an OD of 4 was added to the culture medium, as
well as 100 µg/ml cycloheximide. Before fixation the cells were
permeabilized with streptolysin O and fixed as described (Krijnse
Locker et al., 1994a). The cells were prepared for
cryosectioning and subsequently double-labeled as described (Slot
et al., 1988, 1991).
Quantitation of the Labeled Bands
Each gradient
fraction was subjected to immunoprecipitation and the labeled proteins
were separated in 15% SDS-PAGE. The gel was fixed for 1 h in 20%
methanol, 7% acetic acid and dried. The quantitation of the bands was
performed by PhosphorImager. In the first fraction from the bottom of
the gradient as well as in the pellet we consistently observed a
labeled band of approximately 25 kDa, most likely derived from VV, that
almost co-migrated with the Mform of the M protein. We
therefore quantitated the 25-kDa protein in the pellet fraction and
substracted its value from that of the first fraction of the gradient.
Intracellular Localization of the MHV-M
Protein
We have recently shown that the M protein, when
expressed using a vaccinia virus recombinant localized to late Golgi
and the TGN (Krijnse Locker et al., 1992a). Moreover, we
observed that the M protein, unlike TGN38 and furin (Humphrey et
al., 1993; Bos et al., 1993; Molloy et al.,
1994), does not appear to recycle between the plasma membrane and the
TGN (Krijnse Locker et al., 1994b). For the present study we
wanted to determine in more detail how much of the M protein would
reach the TGN after a 2-h chase with cycloheximide. For this we made
use of a HeLa cell line stably transfected with the human -2,6
sialyltransferase tagged on its COOH terminus (the luminal side) with
the P5D4 epitope of the VSV-G protein.
(
)
These
cells were infected with the recombinant VV expressing the M protein.
Six hours after infection we treated the cells for 2 h with the
endocytic marker bovine serum albumin-gold in the presence of
cycloheximide. Under these conditions, by blocking protein synthesis we
chased the M protein to its final location, whereas the cells
internalized the gold to essentially all endocytic compartments. The
intracellular distribution of the M protein was determined using the
P5D4-tagged sialyltransferase as a marker of the TGN (Roth et
al., 1985). In most of the cells the M protein was clearly located
on one side of the stack, co-localizing in part with sialyltransferase
in the TGN (Fig. 2). Only very little label for the M protein was
found in endosomes that were labeled with the internalized gold (not
shown). In some cells considerable amounts of the M protein were also
found throughout the Golgi stack extending beyond the label for
sialyltransferase, probably due to accumulation of the protein as a
result of overexpression. These data confirm earlier results showing
that the MHV-M protein localizes to the trans side of the Golgi stack
and to the TGN. They show in addition that under our experimental
conditions we were unable to chase the M protein quantitatively into
the TGN (see below).
Figure 2:
Localization of the MHV-M protein in SA:48
cells. The M protein was expressed using vvMHV-M. The infected cells
were treated with bovine serum albumin-gold and cycloheximide to
``chase'' the M protein. Before fixation the cells were
permeabilized with streptolysin O. The M protein (10 nm gold,
arrowhead) localizes mainly to one side of the Golgi, where it
co-localizes with sialyltransferase (detected by anti-P5D4, 5 nm gold,
small arrow) in the TGN. On this side of the stack typical
electron-dense clathrin coats are visible ( c), as well as
tubular elements ( T). E, endosome. Bars: 100
nm.
The M Protein Occurs in Heterogeneous Complexes in the
Golgi Complex
In MHV infected murine cells the two viral
membrane glycoproteins, M and the spike (S), interact. This interaction
can be followed by co-immunoprecipitation under specific detergent
conditions (Opstelten et al., 1994a). When analyzed in sucrose
gradients the complexes of the M and S proteins appeared to consist of
large heterogeneous heterooligomers of M and S (Opstelten et
al., 1994b). Since the S protein, when expressed independently as
well as in infected cells, is assembled into trimers (Delmas and Laude,
1990)(
)
it seemed likely that the M protein was
responsible for this multimer formation. We decided, therefore, to
analyze the oligomeric structure of the M protein in more detail.
; see Fig. 1) labeled during the
pulse, stayed at the top of the gradient (in the upper four fractions).
However, the glycosylated Golgi forms, formed during the 60-min chase,
were heterogeneously distributed over the gradient (Fig. 3 A).
This heterogeneous pattern did not change after a 120-min chase or
after chasing for up to 6 h (not shown). It also remained unchanged
when intact, or streptolysin O permeabilized cells were treated with
the cross-linker dithiobis(succinimidylpropionate), a
membrane-permeable cross-linker, before lysis (not shown). It is
significant to note in Fig. 3 A that the residual fraction of
the M protein that did not acquire Golgi-specific modifications during
the chase (M
and M
) remained in the upper
fractions of the gradient. To show the M
and M
forms more clearly, an overexposed autoradiogram of the chase is
shown in Fig. 3 B.
Figure 3:
A,
pulse-chase analysis of the M protein. The M protein was expressed by
vvMHV-M in COS cells. The cells were pulse labeled for 12 min and
chased for 60 min. Pulse-labeled and chased cells were lysed and
lysates were layered on top of a 15-30% continuous sucrose
gradient. After centrifugation the gradients were fractionated from the
bottom ( fraction 1) to the top ( fraction 10). The
bottom ( B) of the tube was resuspended in lysis buffer to
dissolve pelleted material. The M protein was immunoprecipitated from
each fraction and analyzed in a 15% PAGE. After the pulse labeling only
the unglycosylated ER form (M; indicated) is apparent at
the top of the gradient. After the chase the Golgi (M
;
indicated) and the TGN (not indicated) forms are observed and these are
heterogeneously distributed over the gradient. In B the chase
part of A has been overexposed to emphasize the M
and M
forms (indicated) that remain at the top of the
gradient.
When testing the effect of
different detergents, CHAPS (20 m
M) appeared to give a similar
heterogeneous distribution of the glycosylated forms, whereas non-ionic
detergents such as Triton X-100 and Nonidet P-40 failed to preserve any
complexes (not shown).
form of the MHV-M protein. Since
some of this 25-kDa protein was apparently also present in fraction
number 1, we quantitated the 25-kDa protein in the bottom fraction and
substracted this amount from that of the first fraction (see
Fig. 3
and ``Materials and Methods''). In order to
estimate the size of the M protein complexes, two proteins with known
s
value were used as
sedimentation markers. From Fig. 4it is clear that the largest
complexes of the glycosylated M protein migrated with sedimentation
values larger than 19 s
;
the largest complexes near the bottom of the gradient migrated around
30 s
. Assuming that these
multimers consist of the M protein only (see ``Discussion'')
the complexes in the first fraction of the gradient corresponded to
approximately 1000 kDa, equivalent to about 40 molecules of the M
protein. Collectively these data indicate the M protein occurs in
heterogeneous complexes in the Golgi complex.
Figure 4:
Distribution of the M protein over the
gradient. The labeled M protein in each gradient fraction of Fig. 3 was
quantitated by PhosphorImager analysis. The amount of radioactivity in
each fraction was plotted as a percentage of the total counts of the M
protein present in all fractions of the gradient. The sedimentation of
two marker proteins catalase (11.3 s) and
thyroglobin (19.3 s
) is indicated. ⊡,
pulse;
, chase.
The VSV-G Protein Does Not Occur in Large
Complexes
The specificity of the above described observations
was investigated by making use of another well characterized viral
membrane protein, the VSV-G protein. This latter protein, like the M
protein, was expressed using a recombinant VV (see ``Materials and
Methods''). The trimerization of the VSV-G protein has been
described in detail, and the preservation of the trimers analyzed using
sucrose gradients requires a low pH (Doms et al., 1987).
Likewise, using the detergent mixture used in this study that requires
a neutral to alkaline pH (Helenius and Simons, 1975), the G protein
remained at the top of the gradient both after the pulse labeling as
well as after the chase, peaking in the second and third fraction from
the top (Fig. 5). It seemed thus that the G protein migrated as a
monomer, both after the pulse and after the chase. Clearly, the protein
was transported through the Golgi complex during the chase, since it
became almost completely endo-- N-acetylglucosaminidase
H-resistant (Fig. 5). When transport to the plasma membrane was
prevented and the G protein was accumulated in the TGN by chasing at 20
°C (Griffiths et al., 1985), no higher order oligomeric
structures were observed under our experimental conditions (not shown).
Figure 5:
Pulse-chase analysis of the VSV-G protein.
COS cells were infected with vvVSV-GtsO45 and pulse-labeled for 30 min
at 39 °C, followed by a chase of 2 h at 32 °C. Cells were lysed
and the G protein analyzed after centrifugation, the same as for Fig.
3. The labeled protein is associated with the upper part of the
gradient in fractions 7 through 10. Part of the lysates was analyzed
without centrifugation; it was aliquoted in two and treated or not
treated with endo-- N-acetylglucosaminidase H ( two
right lanes of the panel).
A Tail-less Mutant of the M Protein That Fails to be
Retained in the Golgi Complex Also Occurs in Complexes
From the
preceding results it is clear that the M protein occurs in
heterogeneous complexes in the Golgi complex, whereas a plasma membrane
protein failed to do so. Therefore, it was of interest to analyze the
behavior of a mutant M protein that fails to be retained in the TGN but
rather is transported to the plasma membrane. Deletion of the 22
COOH-terminal amino acids of the M protein results in the delivery of
the mutant protein to the plasma membrane (Armstrong and Patel, 1991;
Krijnse Locker et al., 1994b).
COOH) was pulse labeled and chased for 3 h to
accumulate more than 90% of the labeled protein at the plasma membrane
as determined by surface immunoprecipitation (Fig. 6). The cells were
lysed and the protein analyzed as before. From Fig. 6it is clear
that the mutant protein behaved similar to the wild-type protein
appearing in heterogeneous complexes that now occurred at the cell
surface. Upon closer inspection, however, the complexes of this
particular protein appeared to be consistently smaller than those of
the wild-type protein, since they were absent from the two lowest
fractions from the bottom. Also, they appeared to be more homogeneous,
since they were largely concentrated in the fractions 4 through 6. This
enabled us to estimate their size in more detail. By running the marker
proteins catalase and thyroglobulin and M
COOH in one gradient the
average size of the mutant protein's complexes appeared to be
about 19 s
corresponding to
about 650 kDa or 30 molecules of the mutant protein.
Figure 6:
Analysis of MCOOH. COS cells infected
with vvMHV-M
COOH were pulse labeled for 30 min and chased for 3 h
(only the chase is shown). The protein was analyzed in a sucrose
gradient and immunoprecipitated from each fraction. The Golgi
(M
; indicated) and the TGN forms (not indicated) are
heterogeneously distributed over the gradient, but only a minor
fraction of the protein is associated with the bottom two fractions, 1
and 2. To estimate the amount of this protein present at the plasma
membrane, a surface immunoprecipitation was carried out. The right
panel shows that most of the protein was at the plasma membrane
( S) and only a small amount of the M
form had
remained intracellularly ( I) after the 3-h
chase.
Determination of the Initial Site of Complex
Formation
From Fig. 3, A and B, it is
clear that the unglycosylated ER form of the M protein (M)
and the M
form, which carries the IC modification
N-acetylgalactosamine (GalNAc; Krijnse Locker et al.,
1994a), did not appear in large complexes. Also, when the M protein was
labeled continuously for 2 h the M
and M
species were not found in complexes (not shown). This suggests
that complex formation of the M protein occurs upon, or after, arrival
in the Golgi complex. In an attempt to determine where in the Golgi
complex, the complexes would first be detected, we labeled the M
protein in the presence of BFA, thereby preventing it from reaching the
TGN (Chege and Pfeffer, 1990; Krijnse Locker et al., 1992a).
and M
form after the
pulse. After the chase only the M
form was made, whereas
the TGN forms (M
and M
) were not apparent
(Fig. 7; see also Krijnse Locker et al., 1992a). From
Fig. 7
it is clear that in the presence of BFA the M protein was
not incorporated into complexes and instead migrated as a monomer. In
order to find out whether this lack of complex formation was due to a
general effect of BFA or to the failure of the M protein to reach the
compartment where the large complexes are formed, e.g. the
TGN, we carried out the following experiment. The M protein was
pulse-labeled and chased for 3 h without BFA to enable the bulk of the
protein to reach the Golgi and the TGN. The cells were then treated for
1 h with BFA before lysis and analysis of the M protein in sucrose
gradients. As shown in Fig. 8 the M protein complexes that were formed
before the BFA treatment were partially resistant to the BFA treatment,
since it appeared that less of the M protein was now detected in the
bottom five fractions. Since the distribution of the Golgi and TGN
forms of the M protein over the gradient does not change with
increasing times of chase (see above), the effect of the 1 h BFA
treatment must be due to a partial dissociation of the complexes.
Figure 7:
Analysis of the M protein after BFA
treatment. Infected COS cells were pulse-labeled for 15 min and chased
for 60 min in the presence of 5 µg/ml BFA. The M protein was
analyzed as described in the legend to Fig. 3. In the presence of BFA
only the Mform (indicated) is made during the
chase.
The IBV-M Protein Does Not Form Large Oligomers
We
next analyzed whether the IBV-M protein, a protein that has previously
been localized to the cis-Golgi region (Machamer et
al., 1990; Sodeik et al., 1993; Klumperman et
al., 1994) did appear in large complexes under our conditions.
Previous experiments had shown that the wild-type IBV-M protein does
not form oligomers when using Triton X-100, whereas a chimeric protein
containing the first transmembrane domain of this latter protein did
(Weisz et al., 1993). We wanted to know whether this failure
to detect oligomers of the wild-type IBV-M protein was due to the
experimental conditions used in the latter study. However, consistent
with the results of Weisz et al. (1993), when the IBV-M
protein expressed in COS cells, was sedimented in sucrose gradients
like the MHV-M protein and detected by ECL the bulk of the protein was
found in the first four fractions from the top (Fig. 9) and
apparently failed to form multimers.
Figure 9:
Analysis of the IBV-M protein.
vvIBV-M-infected COS cells were lysed at 9 h post-infection and the
lysate centrifuged as described in the legend to Fig. 3. The proteins
in each fraction were trichloroacetic acid-precipitated and separated
in a 15% SDS-PAGE. The proteins were transferred onto nitrocellulose,
and the IBV-M protein was detected by ECL.
Oligomerization of the MHV-M Protein
The data
from this study show that the newly synthesized M protein of MHV occurs
in large detergent-insoluble complexes following its arrival in the
Golgi complex. Importantly, this complex formation differed in many
respects from the oligomerization that has been extensively described
for the VSV-G and influenza HA protein (Doms et al., 1987;
Copeland et al., 1988). In contrast to the latter process that
occurs in the ER/IC (Doms et al., 1993), the formation of the
M protein complexes appeared to start only after the protein had left
both the rough ER and the IC, as judged by its state of
O-glycosylation. In an attempt to determine where in the Golgi
complex these complexes would occur, we used BFA. In the presence of
this drug the M protein failed to oligomerize, suggesting that the
oligomerization may start in the TGN. However, already formed complexes
seemed to partially dissociate upon BFA treatment. From the EM data
(see Fig. 2) and from previous data (Krijnse Locker et
al., 1992a; Krijnse Locker et al., 1994b), it seems clear
that after a 3-h chase period the M localizes to the stack as well as
to the TGN. Since newly synthesized M does not form complexes in the
presence of BFA, this result may suggest that the M oligomers that are
in the Golgi stack after the chase dissociate upon subsequent BFA
addition, whereas the complexes in the TGN may resist the BFA
treatment. This may imply that the complex formation starts in the
Golgi and persists in the TGN. Alternatively, the drug may destabilize
the complexes, and their partial dissociation caused by BFA, may be
nonspecific and not related to their intracellular location.
Comparison to the Oligomerization of the IBV-M
Chimera
The formation of detergent insoluble complexes has been
described before for a Golgi-resident protein. A chimeric construct
consisting of the VSV-G protein that had its anchor domain replaced by
the first transmembrane domain of the IBV-M protein (called Gm1; Swift
and Machamer, 1991) has been shown to form Triton X-100-insoluble
complexes upon arrival in the Golgi complex (Weisz et al.,
1993). It is not clear, however, whether these observations are
comparable with ours. First, the IBV-M complexes were found for the
chimeric construct only and not for the wild-type IBV-M protein,
whereas the MHV-M complexes were obtained with the wild-type protein as
well as with some of our mutant M proteins (see below). Moreover, under
our experimental conditions we were unable to detect complexes of the
wild-type IBV-M protein. Second, the complexes of Gm1 were stable in
Triton X-100, a detergent that failed to preserve MHV-M complexes. It
is also not clear whether the Gm1 complexes described by Weisz et
al. (1993) were heterogeneous as are the MHV-M complexes.
Moreover, the different analytical conditions (5-20% versus 15-30% sucrose gradients) do not allow direct comparison.
The Gm1 oligomers appeared to be smaller, consisting of approximately
12 molecules of the chimera, whereas the largest oligomers of the MHV-M
protein contained at least 40 molecules. Finally, the MHV-M complexes
persisted at the plasma membrane, whereas oligomers of the Gm1 protein
were no longer observed with mutants that were transported to the cell
surface (Weisz et al., 1993). It seems, therefore, that the
phenomenon of complex formation as observed for Gm1 and for MHV-M
reflects different cellular processes.
Detergent Insolubility of Other Membrane
Proteins
The phenomenon of detergent insolubility of membrane
proteins destined for the plasma membrane has been documented before.
Thus, the influenza virus HA protein has been shown to become partially
detergent-insoluble in Triton X-100 and CHAPS (Skibbens et
al., 1989; Kurzchalia et al., 1992; Fiedler et
al., 1993). When the insoluble complexes of the HA protein that
are seen with both these detergents were compared, it appeared that
they were qualitatively similar but the Triton X-100 complexes were
richer in (glyco)lipids (more than 95% (w/w) lipid content) than those
formed in CHAPS (40%, w/w). Based on these observations it was
concluded that the CHAPS complexes may be dominated by protein-protein
interactions (Fiedler et al., 1993). In our study the MHV-M
protein complexes were not preserved in Triton X-100 but were formed in
the presence of both CHAPS and the sodium deoxycholic acid/Nonidet P-40
mixture. It is, therefore, tempting to speculate that the MHV-M
complexes are also relatively more enriched in protein rather than
lipids (see below). This view is consistent with our finding that the M
protein complexes do not flotate in sucrose gradients (not shown).
Retention Mechanism of the MHV-M Protein
We and
others (Armstrong and Patel, 1991; Krijnse Locker et al.,
1994b) have shown previously that deletion of the cytoplasmic tail of
the MHV-M protein resulted in transport to the plasma membrane of the
mutant protein. This tail has features of an internalization signal,
but unlike TGN38 or furin (Humphrey et al., 1993; Bos et
al., 1993; Molloy et al., 1994) the MHV-M does not seem
to recycle from a distal compartment. The cytoplasmic tail alone,
however, was not sufficient for TGN retention; mutant MHV-M proteins
that still contained the tail but lacked one or two transmembrane
domains were now detected in endosomes, but were not subjected to rapid
lysosomal degradation. The amphiphilic cytoplasmic tail, except for the
last 22 amino acids, appeared to have no information for Golgi
retention, since deletion of this part resulted in a mutant protein
that localized in an identical fashion to the wild-type protein (late
Golgi and TGN). The same appears to apply for the luminal amino
terminus, since deletion of this part did not affect its retention in
the Golgi and the TGN. Based on these results we have concluded that
the retention of the MHV-M protein is determined by two components, one
contained in the transmembrane domains and one in the cytoplasmic tail
(Krijnse Locker et al., 1994b). When some of these mutant
proteins were analyzed using sucrose gradients, we found that deletions
in the cytoplasmic tail (deleting either the extreme 22 COOH-terminal
amino acids or the complete cytosolic tail except for these 22 amino
acids) or deletion of the 25 NH-terminal residues, resulted
in complex formation that was similar to the wild-type protein (see
e.g. Fig. 7
; not shown). However, mutant proteins with
only one or two transmembrane domains showed significantly less or no
oligomerization (not shown). This strongly suggests that the presence
of all three transmembrane domains is required for the formation of the
multimers.
(
)
Our data, therefore, clearly differ
from that of Weisz et al. (1993) and Schweizer et al. (1994) that suggested that oligomerization alone is sufficient for
retention. They also differ from the recent results on TGN38 that
showed that either the transmembrane domain or the cytoplasmic tail
themselves were sufficient for retention (Ponnambalam et al.,
1994). It is interesting to note, however, that the complexes of the
mutant M protein, that is transported to the plasma membrane, were
clearly smaller than those of the wild-type protein. We have estimated
their size to be around 650 kDa or 30 molecules. Perhaps a complex of
this size might not be excluded from a 50-100-nm transport
vesicle en route to the plasma membrane (Nilsson et al.,
1994).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.