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From the
Recent studies on proteins residing in the Golgi complex
revealed that the membrane-spanning domain of these proteins are
largely responsible for their retention in the Golgi complex. We show
here that
Newly synthesized proteins are either released into the
cytoplasm or, if they contain a signal peptide, are translocated into
the ER,
The Golgi complex plays a central role
in intracellular sorting and transport (Palade, 1975). The Golgi
complex is also the site glycosylation of nascent proteins takes place;
there are over 100 different glycosyltransferases involved in the
synthesis of protein- and lipid-bound oligosaccharides (Schachter and
Roseman, 1980; Beyer and Hill, 1982). While newly synthesized proteins
pass through the Golgi, these enzymes are retained in the Golgi complex
against membrane ``bulk flow.'' The mechanism underlying such
an apparent immobilization of glycosyltransferases in the Golgi
membranes has not been well understood.
Galactosyltransferase (GT;
UDP-galactose:
GT is a type II integral
membrane protein (Masri et al., 1988; Shaper et al.,
1988; Nakazawa et al., 1988; D'Agostaro et al.,
1989), as are all the Golgi glycosyltransferases cloned to date; they
contain a short amino-terminal cytoplasmic tail, a non-cleavable
signal-anchor/transmembrane domain, a lumenal stem region, and a large
carboxyl-terminal catalytic domain exposed to the Golgi lumen (Paulson
and Colley, 1989).
Recent studies have shown that sequences within
and adjacent to the transmembrane domain of these Golgi enzymes specify
Golgi retention (Munro, 1991; Nilsson et al., 1991; Aoki
et al., 1992; Burke et al., 1992; Colley et
al., 1992; Russo et al., 1992; Tang et al.,
1992; Teasdale et al., 1992; Wong et al., 1992).
Importance of membrane-spanning domains for Golgi retention has been
shown in virus envelope proteins which are also retained in the Golgi
(Machamer and Rose, 1987; Swift and Machamer, 1991). There is, however,
no amino acid sequence similarities in the transmembrane domain among
these Golgi proteins. Because of the lack of a common amino acid
sequence within and/or adjacent to the transmembrane domain in these
Golgi proteins, it is difficult to predict a peptide sequence for a
Golgi retention signal. This is in contrast to that of ER resident
proteins, which are distinguished by the presence of a
carboxyl-terminal tetrapeptide. Proteins bearing this signal can be
retrieved from the Golgi complex back to the ER by the receptor that
recognizes this signal motif (Lewis and Pelham, 1992). It is not known,
however, if the proteins residing in the Golgi are recycled in a
similar retrieving machinery.
In this report, we show data
suggesting that GTs are forming homodimers in vivo, and such
homodimerization is largely dependent on Cys
For digestion
with endo-
When the immunoprecipitates using anti-TfR
antibodies were analyzed by SDS-PAGE (Fig. 5, lanes
1-3), monomer and dimer of GT
These results (Fig. 5) indicate that,
regardless of the antibodies or reporter molecules used,
co-precipitation of 50- and 52-kDa proteins depends on the portion of
GT molecule consisted with cytoplasmic tail, transmembrane domain, and
stem region.
COS-1 cells were transfected with
GT
In
this paper, we investigate the molecular mechanism of Golgi retention
by using GT as a model system. We found that the double point mutation
(Cys
Our
co-immunoprecipitation experiments demonstrated that GT oligomers
associate with
Presently, we do not know how GTs
interact with
Currently, two models
have been proposed for the mechanism of Golgi retention, which are not
mutually exclusive. (i) Golgi enzymes form large hetero- and/or
homo-oligomers via their lumenal and transmembrane domains by
``kin recognition.'' The oligomers attach to the matrix via
the cytoplasmic domains, leading to prevent Golgi enzymes from entering
into the budding transportation vesicles (Weisz et al., 1993;
Nilsson et al., 1994; Slusarewicz et al., 1994). (ii)
Retention depends on the length of transmembrane domains and the lipid
composition of the membranes in the Golgi complex; increasing the
length of the transmembrane domain could direct Golgi enzymes to the
plasma membrane (Munro, 1991; Masibay et al., 1993; Bretscher
and Munro, 1993; Pelham and Munro, 1993).
The results obtained by
present study are consistent with the first model, in which
oligomerization of proteins is an important factor for being retained
in the Golgi complex. The present study also suggests that GT oligomers
are stabilized in the Golgi by their direct or indirect association
with tubulins and that the Golgi retention is a part of the
intracellular process controlled by the cytoskeleton.
We thank Drs. Ian S. Trowbridge, John K. Rose, and
Hisashi Narimatsu for their gifts of antibodies and cDNAs. We also
thank Dr. Kazuo Fujikawa (University of Washington, Seatle, WA) for
amino acid sequencing; Drs. Harry Schachter (The Hospital for Sick
Children, Toronto, Canada), Minoru Fukuda, and Yu Yamaguchi (La Jolla
Cancer Research Foundation) for their critical reading of the
manuscript; and Scott Armstrong for technical assistance.
Volume 270,
Number 20,
Issue of May 19, pp. 12170-12176, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-1,4-Galactosyltransferase
MEMBRANE-SPANNING DOMAIN-DEPENDENT HOMODIMERIZATION AND ASSOCIATION
WITH 
- AND 
-TUBULINS (*)
-1,4-galactosyltransferase (GT) forms homodimers and
large oligomers in vivo, and the formation of the homodimers
is dependent on cysteine and histidine residues within the
transmembrane domain. Double mutations of these residues, Cys
Ser and His
Leu, abolish
homodimerization and simultaneously reduce the Golgi retention.
Co-immunoprecipitation of GT and various GT chimeras with anti-GT and
anti-reporter molecule antibodies revealed that large aggregates of GT
are associated with
- and -tubulins and also with other
cellular proteins. This association between tubulins and GT suggests a
supportive role of the cytoskeleton in the Golgi retention mechanism.
()
entering into the sorting pathway
(Walter and Lingappa, 1986). Intracellular transport of proteins from
the ER to plasma membranes is mediated by a series of carrier vesicles
that bud from one compartment and then fuse with the next, giving rise
to vectorial movement (Rothman and Orci, 1992). This transport is
believed to occur by ``default,'' meaning that proteins which
do not have either a signal for transport to a particular organelle or
a specific retention signal are transported to plasma membranes
(Pfeffer and Rothman, 1987).
-D-N-acetylglucosaminide
-1,4-galactosyltransferase, EC 2.4.1.22) is one of the best known
glycosyltransferases and catalyzes the transfer of galactose from
UDP-galactose to N-acetylglucosamine, forming the
Gal-1,4-GlcNAc linkage present in glycoproteins, glycolipids and
proteoglycans (Beyer and Hill, 1982). The biosynthesis of GT showed
that GT is retained in the Golgi with a half-life for 21 h and is also
transported to plasma membranes in HeLa cells (Strous and Berger, 1982;
Strous, 1986). The localization of GT in the trans-cisternae
of the Golgi complex has been well documented by immunoelectron
microscopy (Roth and Berger, 1982). In some types of cells, GT is also
present in plasma membranes (Lopez et al., 1985; Roth et
al., 1985; Bayna et al., 1988; Suganuma et al.,
1991). The association of GT with the cytoskeleton has been suggested
in some reports (Eckstein and Shur, 1992; Strous et al.,
1991), although the significance of such observations in relation to
the Golgi retention has not been explored.
and
His
residues in the transmembrane domain. Mutation of
these two amino acid residues significantly affect the retention of GT
in the Golgi complex. Furthermore, we show that
- and
-tubulins are associated with oligomers of GT, suggesting that
such an interaction may be an important factor for Golgi retention
mechanism.
Chimeric Constructs
cDNAs encoding human CG
(Fiddes and Goodman, 1979) and TfR (Schneider et al., 1984)
were kindly provided by Drs. J. Rose (Yale University, New Haven, CT)
and I. Trowbridge (The Salk Institute, La Jolla, CA), respectively.
cDNAs encoding human GT (Masri et al., 1988), double-point
mutated GT, GT tagged with the C-terminal half of human CG, and
human TfR inserted into the eukaryotic expression vector pcDNAI
(Invitrogen, San Diego, CA) have been described previously (Aoki et
al., 1990, 1992). Introduction of RsrII and XhoI
sites into the CG cDNA generated a fragment of the cDNA encoding
the mature CG protein lacking a signal sequence. This fragment was
ligated at the RsrII and XhoI sites in pcDNAI
containing GT and to a corresponding fragment containing double-point
mutated GT cDNAs, resulting in GTCG
and
GTCG
, respectively (see Fig. 1).
GTTfR and GT
TfR were
constructed in the same manner as GT
CG by introducing
RsrII and XbaI sites into the extracellular domain of
TfR (amino acids 97-760).
Figure 1:
Schematic diagram
of human GT and TfR and their chimeric proteins. SL mutants represent a
double point mutation in the GT transmembrane domain, Cys
Ser and His
Leu. CG
,
human chorionic gonadotropin subunit (Fiddes and Goodman, 1979);
C, cysteine; H, histidine; S, serine;
L, leucine; CYT, cytoplasmic tail; TM,
transmembrane domain; STEM, stem region; EX,
extracellular region; CATALYTIC, catalytic domain. The
numbers refer to the amino acid sequence in the wild-type
protein.
Antibodies
Rabbit anti-human CG antibodies,
fluorescein isothiocyanate-conjugated F(ab`)
fragments of
goat anti-rabbit IgG antibodies, and fluorescein
isothiocyanate-conjugated F(ab`) fragments of goat
anti-mouse IgG antibodies were obtained from Cappel Laboratories. Mouse
monoclonal anti-human TfR antibody (B3/25) (Trowbridge and Omary, 1981)
and mouse monoclonal anti-human GT antibody (#8626) (Uemura et
al., 1992) were kindly provided by Drs. I. Trowbridge (The Salk
Institute, La Jolla, CA) and H. Narimatsu (Soka University, Tokyo,
Japan), respectively. Rabbit anti-human GT antiserum was prepared by
immunizing a rabbit with soluble form of GT, which was purified to
homogeneity by N-acetylglucosamine-Sepharose affinity
chromatography column from human milk, according to the established
method (Barker et al., 1972).
Transfection and Immunostaining
COS-1 cells were
transfected with a cytomegalovirus promoter-driven pcDNAI vectors
containing chimeric constructs using DEAE-dextran (Aoki et
al., 1992) or DOTAP transfection reagent (Boehringer Mannheim),
according to the manufacturer's instruction. For preparation of a
stably transfected cell line, CHO cells co-transfected with
GT1/2CG
and pSVNeo were selected in the
presence of 500 µg/ml Geneticin (Life Technologies, Inc.).
Resistant clones were isolated and cloned by limiting dilution for
selection of cloned cell lines. Expression of the cDNA products was
examined by immunofluorescence staining of transfected cells using
anti-CG antibodies, as described (Aoki et al., 1992).
Merged confocal images were obtained using an MRC-600 Laser-scanning
Confocal Imaging system (Bio-Rad).
Immunoprecipitation
COS-1 cells were transfected
by plasmid vectors having cDNAs encoding GT, TfR, or chimeric proteins.
On day 2 after transfection, cells were first incubated for 1 h in
methionine-, cysteine-deficient Dulbecco's modified Eagle's
medium supplemented with 5% dialyzed fetal calf serum, then
metabolically labeled for indicated times with
[S]methionine and
[
S]cysteine (100 µCi/ml
Tran
S-label (ICN Radiochemicals), and were chased with
complete Dulbecco's modified Eagle's medium. For long term
labeling, cells were labeled with 100 µCi/ml
Tran
S-label in the presence of 10 µM
methionine and 10 µM cysteine. After 12 h labeling, cells
were lysed with lysis buffer (25 mM HEPES, pH 7.4, 1% Nonidet
P-40, 10% glycerol, 150 mM NaCl, 0.225 TIU/ml aprotinin, 50
µg/ml leupeptin, 10 µg/ml pepstatin A, 2 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, 0.05%
NaN
). Lysates were preadsorbed with protein G-Sepharose
beads (Pharmacia Biotech Inc.) and then reacted with protein
G-Sepharose beads precoated with antibodies for 4 h at 4 °C. The
beads were washed four times at 4 °C with the washing buffer A (25
mM HEPES, pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40,
0.2% sodium deoxycholate, 0.1% SDS), three times at 4 °C with the
washing buffer B (10 mM phosphate buffer, pH 7.4, 500
mM NaCl, 0.5% Nonidet P-40), and once at 4 °C with the
washing buffer A, and then boiled for 3 min in SDS-PAGE sample buffer.
Eluates from the beads were resolved in SDS-PAGE (Laemmli, 1970), and
the immunoprecipitates were detected by fluorography.
-N-acetylglucosaminidase H (endo H), the
immunoprecipitates were eluted with 20 mM Tris-HCl buffer, pH
7.5 containing 0.15 M NaCl, 1% SDS, and 10 mM
dithiothreitol, diluted with 9 volumes of 0.15 M sodium
citrate, pH 5.3, containing 0.5 mM phenylmethylsulfonyl
fluoride, and then incubated at 37 °C for 18 h in the presence or
absence of 100 milliunits/ml endo H (Boehringer Mannheim). Following
trichloroacetic acid precipitation, the proteins were dissolved in SDS
sample buffer, electrophoresed, and analyzed by a BAS 2000 radioactive
imager (Fuji Film).
Purification and Amino Acid Sequencing of GT-associated
Proteins
CHO cells stably transfected with
GT1/2CG were cultured in Eagle's medium
supplemented with 5% fetal calf serum. Cells collected from 34 culture
dishes (15-cm diameter) were solubilized with the lysis buffer
described above. After centrifugation, the supernatant was applied to a
protein A-Sepharose CL-4B column for preclearing, and subsequently to a
rabbit anti-human GT antibodies covalently immunoaffinity (Protein A
Sepharose) column, which was prepared as reported (Schneider et
al., 1982). After washing with the washing buffers A and B, bound
proteins were eluted with 0.1 M glycine, pH 2.5, 0.1% Nonidet
P-40. The eluates were immediately neutralized with 1 M Tris,
pH 9, and precipitated with 6% trichloroacetic acid. The precipitates
were dissolved in SDS-PAGE sample buffer containing 5%
-mercaptoethanol, heated to 50 °C for 20 min, and subjected to
SDS-PAGE, followed by electrotransfer onto a PVDF membrane (Matsudaira,
1987). After staining with Coomassie Blue, the bands corresponding to
50 and 52 kDa were excised, and the amino-terminal sequences of the
proteins were determined on an Applied Biosystems 470A gas-phase
sequencer equipped with an on-line phenylthiohydantoin-amino acid
analyzer. The analysis was kindly performed by Dr. Kazuo Fujikawa at
the University of Washington.
Sucrose Density Gradient Sedimentation
Continuous
5-20% (w/w) sucrose gradients were poured over a 60% (w/w)
sucrose cushion, essentially as described (Zagouras et al.,
1991), except that all solutions were in 25 mM HEPES, pH 7.4,
1% Nonidet P-40, 150 mM NaCl, 0.05% NaN. COS-1
cells expressing chimeric proteins were metabolically labeled and lysed
with the lysis buffer as described above. Lysates were loaded on top of
the gradients and centrifuged at 38,000 rpm for 19 h at 4 °C.
Fractions (0.8 ml) were collected from the top, immunoprecipitated, and
electrophoresed followed by fluorography.
RESULTS
Golgi Retention of GT Chimeric Proteins in COS-1
Cells
In our previous studies, the expression of GT chimeras was
examined 2 days after transfection by immunofluorescence microscopy,
and a double point mutation, Cys
Ser and His
Leu, within the GT transmembrane domain has been shown to
profoundly affect Golgi retention (Aoki et al., 1992). The
expression of GT
1/2CG protein on cell surface
was demonstrated by immuno-staining of intact COS-1 cells followed by
fluorescence-activated cell sorter analysis (Aoki et al.,
1992). Localization of several different chimeric proteins consisted
with the portion of GT (the cytoplasmic tail, the transmembrane domain,
and the stem region) and with different reporter molecules (whole
CG
or the extracellular domain of TfR, see Fig. 1) were
tested for their Golgi localization in transfected COS-1 cells by
immunofluorescence microscopy. GTCG and
GT
TfR proteins were mainly localized in the Golgi
complex on 2 or 3 days after transfection (see Fig. 2B for GT
TfR). Double point mutation (Cys
Ser and His
Leu) of both these
chimeric proteins resulted in diffuse distribution (see Fig. 2,
C and D for GT
TfR), including cell
surface (data not shown; see Aoki et al. (1992)). On the other
hand TfR
were localized on the cell surface
(Fig. 2A), although there was also some staining of the
cell interior, including the Golgi complex.
Figure 2:
Confocal microscopy of localization of
TfR, GT
TfR, and
GT
TfR proteins in COS-1 cells. COS-1 cells were
transiently transfected with TfR
(A),
GT
TfR (B), or GT
TfR
(C and D). On day 2 after transfection, cells were
fixed and permeabilized with saponin. TfR
proteins and
chimeric proteins were stained with monoclonal anti-TfR antibodies
followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG
antibodies. The merged pictures of 10-15 Z (tangential) sections
of the cells are shown in A (surface expression), B (Golgi localization), and C and D (delocalized
expression). In D, the multinucleated cell shows delocalized
expression of GT
TfR proteins at the left.
Simultaneously the cell showing Golgi localization of the proteins is
observed at the right of the same
field.
Pulse-labeling and chase
experiments (Fig. 3) showed that at chase time 0 all of the
GTTfR (lane 1) and
GT
TfR (lane 5) are converted to 75-kDa
protein (lanes 2 and 6) by endo H digestion. After 2
h chase, about 40% of GT
TfR was converted to 75
kDa band (lane4, lowerband),
suggesting that this portion of GT
TfR was remained
in the ER while the rest of the GT
TfR (lane4, upper band) was transported to the Golgi.
After 2 h of chase, about 20% of GT
TfR mutant was
converted to the band of 75 kDa (shown by an arrow in lane8) by endo H, suggesting that this portion of
GT
TfR was remained in the ER while most of
GT
TfR (lane8, upperband) was transported to the Golgi. Thus Cys
and His
residues in the transmembrane domain of GT
appear to affect both intracellular transport and Golgi retention.
Figure 3:
Endo H
digestion of metabolically labeled GTTfR and
GT
TfR
COS-1 cells were transiently transfected with
either GT
TfR or GT
TfR. On day 2
after transfection, cells were pulse-labeled with
[
S]Met and [
]Cys for 30
min and chased in regular culture medium for 0 or 2 h as indicated. The
chimeric proteins were immunoprecipitated with monoclonal anti-TfR
antibodies, and immunoprecipitates were treated with (+) or
without (-) endo H prior to analysis by SDS-PAGE. Numbers on the
left are molecular markers (kDa).
Dimer Formation Depends on Cys
COS-1
cells were transfected with vector alone, plasmid vectors encoding
TfRand
His
in the GT Transmembrane Domain
, GT
TfR, or
GT
TfR (Fig. 1). On day 2 after transfection,
COS-1 cells were labeled with [
S]Met and
[
S]Cys for 1 h, followed by a 2-h chase period,
and were immunoprecipitated using anti-TfR antibodies. When the
immunoprecipitates were analyzed by SDS-PAGE, all mature proteins of
TfR
, GT
TfR, and
GT
TfR were detected as approximately 90-kDa bands
under reducing conditions (Fig. 4). The formation of dimers
(approximately 180 kDa, shown by an arrowhead in lane
3) was observed in the cells transfected with
GT
TfR (lane 3) but not with
GT
TfR (lane 2). These results suggest that
GT
TfR polypeptides are forming homodimers in these
cells, which appear to be relatively resistant to SDS and
-mercaptoethanol. These results also show that the dimer formation
is largely dependent on the presence of Cys and His
residues in the transmembrane domain of GT.
Figure 4:
Fluorogram of SDS-PAGE of
immunoprecipitate. COS-1 cells were transiently transfected with either
vector alone (lane1), TfR (lane2), GT
TfR (lane3), or GT
TfR (lane4). On day 2 after transfection, cells were pulse-labeled
with [
S]Met and [
S]Cys
for 1 h followed by 2 h of chase. Cell lysates were immunoprecipitated
with monoclonal anti-TfR antibodies in the presence of protein
G-Sepharose beads. The beads were washed as described under
``Materials and Methods.'' The immunoprecipitates were boiled
in SDS-PAGE sample buffer in the presence of 5%
-mercaptoethanol,
analyzed by SDS-PAGE on a 6.5% gel, and detected by fluorography.
Molecular markers are shown in kDa.
Because Cys and
His residues are often involved in metal chelating as exemplified in
zinc finger proteins (Beng, 1990), the effect of EDTA and
o-phenanthroline on the dimers were examined. Thus the
immunoprecipitates of GTTfR were boiled with 50
mM EDTA or 10 mMo-phenanthroline in the
presence of SDS and
-mercaptoethanol just prior to SDS-PAGE. EDTA
and o-phenanthroline, however, did not dissociate the dimers
to monomers (data not shown). These observations suggest that metal
chelating is not involved in the dimer formation between GT molecules.
Dimer formation of GT is probably due to the secondary structure
induced by a unique conformation of peptide within the lipid bilayer
(Bormann et al., 1989; Machamer, 1993).
Association of GT with Cellular Proteins
In
parallel to the above described experiments, we examined whether any
proteins are associated with GT. We performed extensive
co-immunoprecipitation analysis using GT and three GT
chimeric proteins, GT
1/2CG,
GT
CG, and GT
TfR (see
Fig. 1
for chimeras). COS-1 cells were transfected with plasmid
vectors containing these cDNAs. On day 2 after transfection, COS-1
cells were labeled with [
S]Met and
[
S]Cys, and cell lysate was subjected for
immunoprecipitation.
TfR were
detected as 90-kDa (lane 3, asterisk) and 180-kDa
(lane 3, arrowhead) bands, respectively.
TfR
, which can pass through the Golgi complex without
apparent Golgi retention, was used as a control. COS-1 cells
transfected with GT
were also included as a control to
detect any nonspecific component co-immunoprecipitated with anti-TfR
antibodies. In these analyses, we repeatedly found that two proteins at
approximately 50 and 52 kDa (Fig. 5, arrows) were
co-immunoprecipitated with the portion of GT molecule that contains the
cytoplasmic tail, transmembrane domain, and stem region. The 50- and
52-kDa proteins were not detected or were only faintly detectable in
the immunoprecipitates of TfR
(lane2) and that of control (lane1).
Figure 5:
Co-immunoprecipitation of GT with cellular
proteins. COS-1 cells were transiently transfected with GT (lane1), TfR
(lane2), GT
TfR (lane3), GT
1/2CG (lane4), GT
(lane5), and
TfR
(lane6). On day 2 after
transfection, cells were labeled with [
S]Met and
[
S]Cys for 21 h. Cell lysates were
immunoprecipitated with monoclonal anti-TfR antibodies (lanes1-3) or monoclonal anti-GT antibodies (lanes
4-6) in the presence of protein G-Sepharose beads. The
immunoprecipitates were analyzed as described in Fig. 4. The positions
of monomers and dimers of each chimeric protein are shown by
asterisks and arrowheads, respectively. Arrows indicate the major co-immunoprecipitated materials with the
proteins possessing the portion of GT structure (see Fig. 1). Molecular
markers are shown in kDa.
COS-1 cells transfected with GT1/2CG,
GT
, and TfR
were subjected for
immunoprecipitation using polyclonal anti-GT antibodies (Fig. 5,
lanes 4-6). GT
1/2CG proteins were
detected at approximately 66, 63, and 57 kDa (monomers;
asterisks in lane4) and at 132, 126, and
114 kDa (dimers; arrowheadsin lane4). The difference in these molecular sizes are most
likely caused by diffferences in glycosylation. GT
was
detected at 52 kDa (monomer; asterisk in lane5) and 104 kDa (dimer; arrowhead in
lane5). The 50- and 52-kDa proteins were detected
again in the immunoprecipitate of GT
1/2CG
(lane4). Although these two co-immunoprecipitated
proteins are not clearly visible in the immunoprecipitates of
GT
(Fig. 5, lane5), this is
because 50- and 52-kDa proteins overlap with GT
proteins on SDS-PAGE. The 50- and 52-kDa proteins were not
detected or only faintly detectable in the control (lane6).
Identification of the 50- and 52-kDa Proteins
To
identify the 50- and 52-kDa proteins that co-immunoprecipitated with
GT, these proteins were purified and their amino acid sequences were
determined. The 50-kDa and 52-kDa proteins were co-purified with GT
from the lysate of CHO cells stably expressing
GT1/2CG by affinity chromatography using an
anti-GT antibody column (see details under ``Materials and
Methods''). The immunopurified materials were separated by
SDS-PAGE, electrotransferred onto a PVDF membrane, and subsequently
subjected for amino-terminal sequence analysis. Fig. 6shows the
Coomassie Blue staining of the gel and the amino-terminal sequences
determined. The 52- and 50-kDa proteins were found identical to
-
and -tubulin, respectively. In addition, amino acid sequence
confirmed the 57-kDa protein as GT.
Figure 6:
Purification of
GT1/2CG and 50 kDa and 52 kDa proteins. CHO cells
stably transfected with GT
1/2CG were solubilized.
Cell lysates were applied onto a protein A-Sepharose column for
preclearing. Bound materials were eluted and analyzed by SDS-PAGE.
Lane 1 shows a Coomassie Blue staining pattern as a control.
Then unbound materials from a protein A-Sepharose column were applied
onto a rabbit anti-GT antibody-conjugated protein A-Sepharose column.
After washing, bound materials were eluted and analyzed by SDS-PAGE.
Lane2 shows the gel stained by Coomassie Blue.
Subsequently, the immunoaffinity-purified materials were subjected to
SDS-PAGE and electrotransferred onto a PVDF membrane. After staining
with Coomassie Blue, the corresponding bands were excised and the
amino-terminal sequences of the proteins were determined. The
determined sequences are illustrated at the right. X indicates no signal, and parentheses indicate the most
likely amino acid residues. Molecular markers are indicated in
kDa.
Association of
Since there were reports describing that the
oligomerization is an important factor for Golgi retention of the
resident proteins (Nilsson et al., 1994; Swift and Machamer,
1991), we examined the relationship between oligomerization of GT and
association of tubulins.
- and -Tubulins with GT Dimers
and OligomersTfR, labeled with [
S]Met
and [
S]Cys, and subsequently solubilized.
Lysates were loaded onto 5-20% sucrose density gradients,
centrifuged, and fractionated into 13 fractions. The sucrose gradient
centrifugation was calibrated using molecular size markers, thus the
fractions which should contain GT monomer and dimer were determined to
be fractions 2/3 and 4/5, respectively.
S-Labeled proteins
in each fraction were subjected to immunoprecipitation using anti-TfR
antibodies, and immunoprecipitates were analyzed by SDS-PAGE. As shown
in Fig. 7, monomers of GT
TfR (approximately
90 kDa) were detected in fraction 2 and later fractions. Dimers of
GT
TfR (approximately 180 kDa) were detected in
fraction 4 and later fractions. The GT polypeptides fractionated in
tubes later than fraction 5 are considered as oligomers larger than
trimers. Therefore the result shown in Fig. 7indicates that a
substantial quantity of GT
TfR polypeptides in the
cell lysate behaved as if they are large oligomers. The result
(Fig. 6) also shows that
- and -tubulins appear only in
fraction 7 and later fractions, suggesting that - and
-tubulins associate only with large oligomers of GT but not with a
monomer.
Figure 7:
Sucrose
density gradient sedimentation of GTTfR. COS-1
cells were transiently transfected with GT
TfR. On
day 2 after transfection, cells were pulse-labeled with
[
S]Met and [
S]Cys for 1 h
followed by 2 h of chase. Cell lysates were subjected to
sucrose-density gradient centrifugation. The gradient was split into 13
equal volume fractions. GT
TfR was immunoprecipited
and analyzed by SDS-PAGE followed by fluorography. Twolongarrows indicate
- and -tubulins.
A shortarrow and arrowhead indicate monomer
and dimer of GT. Fractions that are expected to contain GT monomer,
dimer, and oligomers (larger than trimer) are indicated as
mono, di, and oligo, respectively. Molecular
markers are shown in kDa.
DISCUSSION
Recent studies indicate that the transmembrane domain and
adjacent cytoplasmic and luminal domains of Golgi resident proteins are
largely responsible for these proteins to be retained in the Golgi
complex (Machamer and Rose, 1987; Munro, 1991; Nilsson et al.,
1991; Swift and Machamer, 1991; Aoki et al., 1992; Burke
et al., 1992; Colley et al., 1992; Russo et
al., 1992; Tang et al., 1992; Teasdale et al.,
1992; Wong et al., 1992). However, it is not yet clear how
these transmembrane and adjacent domains lead to Golgi retention.
Ser and His
Leu) within
the transmembrane domain of GT, reduced Golgi retention of the molecule
significantly (Fig. 2; see also Aoki et al. (1992)). As
shown in Fig. 4, the presence of these two amino acid residues is
critical for dimer formation. The formation of homodimers may
facilitate further oligomerization of GT (Fig. 7), which could
result in apparent immobilization of GT in the Golgi membrane.
- and -tubulins (Fig. 7). Microtubules,
composed of filamentous polymeric assemblies of a heterodimer of one
- and one -tubulin polypeptides, are cytoskeletal components
of cells and play a central role in rapid organelle movements, which
occur in such processes as vesicle fusion and transport in some vesicle
fusion events, and in some steps of transport (Allen et al.,
1982; Lippincott-Schwartz et al., 1990). In addition,
microtubules are involved in maintaining the Golgi structure since
disruption of the microtubule network during mitosis or drug-induced
disassembly leads to fragmentation and scattering of Golgi complex
throughout the cell (Turner and Tartakoff, 1989; Kreis, 1990; Iida and
Shibata, 1991). Reassembly of the microtubule network results in
reaggregation of the Golgi complex at the microtubule-organizing center
(Ho et al., 1989). Microtubules are the only cytoskeletal
elements for which structural and some functional relationships to the
Golgi complex have been established. Accordingly, it is possible that
- and -tubulins actively participate in the organization of
the Golgi complex, thus playing a role in retention of resident
proteins in the Golgi complex.
- and -tubulins. We envisage that GTs form
homodimers (Fig. 4, 5, and 7), which subsequently further
aggregate to form a large complex in the Golgi membrane (Fig. 7).
Such a complex may associate with proteins present in the cytoplasm, in
the membrane, and in the lumen of the Golgi. As shown in this study,
the dimer formation and subsequent oligomerization may be mediated
primarily by the transmembrane domain. The GT oligomers may then anchor
either directly or indirectly to the bundle of tubulins. As tubulins
are a part of microtubule-based motor proteins, Golgi retention and
transport of GT could be supported and guided by a microtubule-based
network as has been suggested previously (Strous et al.,
1991). Because tubulins localize in the cytoplasm, the association of
GT with tubulins may be mediated by the cytoplasmic tail of GT.
Deletion of the cytoplasmic tail caused leakage of GT to the plasma
membranes (Nilsson et al., 1991; Aoki et al., 1992),
supporting the hypothesis that Golgi retention of GT is stabilized by
its association with cytoplasmic component. However, association of
tubulin may not be the cause of Golgi retention but rather the result
of it, because GT and tubulin are co-localized in HeLa cells, which
were treated with brefeldin A (Strous et al., 1991).
Furthermore, our preliminary data showed that GT mutant
proteins were also associated with
- and -tubulins, although
careful analysis should be carried out to define differences, if any
are present, between GT proteins at the Golgi and at cell
surface. It is possible that other proteins are involved in the
association between GT and tubulins. In fact, we have detected five
additional proteins co-immunoprecipitated with GT and GT chimeras but
not with TfR.(
) It is not presently clear whether
or not other Golgi enzymes also associate with tubulins. Slusarewicz
et al.(1994) identified a functional medial Golgi
``matrix,'' which could promote binding of medial Golgi
enzymes. The matrix includes several proteins, although they appear not
to include - and - tubulins, and GT did not bind to the
matrix. It is therefore possible that the association of tubulins is
unique to GT or trans-Golgi enzymes.
-D-N-acetylglucosaminide
-1,4-galactosyltransferase; TfR, transferrin receptor; CHO,
Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; endo
H, endo--N-acetylglucosaminidase H; PVDF, polyvinylidene
difluoride.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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