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INTRODUCTION |
The Golgi complex plays a crucial role in intracellular transport
and modification of exportable proteins and lysosomal proteins. It is
organized by three functionally distinct subcompartments, the
cis-Golgi network, the Golgi stack, and the
trans-Golgi network (1). During transport through the Golgi
complex, exportable proteins are sequentially modified by processing of
N- and O-linked oligosaccharides (2), proteolytic
cleavage (3), sulfation (4), and so on. These modifications are
accomplished by enzymes localizing on the luminal side of the specified subcompartments.
Localization of these Golgi enzymes has been thought to be
signal-dependent. Type II membrane proteins with a short
cytoplasmic tail, including glycosyltransferases, contain the Golgi
retention signals in and around their transmembrane domains
(TMDs)1 (for review see Ref.
5). Two models have been proposed for how the TMDs of Golgi enzymes
could participate in their Golgi retention; the oligomerization or kin
recognition model (6, 7) and the lipid-sorting or bilayer thickness
model (5, 8). In contrast, the Golgi localization signals of type I
membrane proteins are probably contained in the cytoplasmic tail, as
demonstrated for TGN38 (9) and the proprotein processing enzyme furin
(10, 11), both of which are localized to the trans-Golgi
network by tyrosine-containing motifs. These proteins, irrespective of type I or II, have the common feature in the membrane topology that the
majority of their protein masses protrude from the membrane into the lumen.
Giantin is an integral Golgi-resident protein (12) that has no
cleavable signal sequence at the NH2 terminus but contains a single hydrophobic sequence at the COOH terminus that could participate in membrane localization (13-15). Thus, it is believed that giantin is cytoplasmically oriented and anchored to the Golgi membrane by the COOH-terminal hydrophobic domain (CMD for
"COOH-terminal membrane-anchoring domain"). Although containing 24 amino acid residues (enough for spanning the lipid bilayer of
membranes), the CMD is not considered to be a typical TMD, because
giantin has no luminal side sequence following the CMD. Linstedt
et al. (16) recently demonstrated that newly synthesized
giantin was first inserted into the endoplasmic reticulum (ER) membrane
and then transported to the Golgi complex in the same manner as is well
known for Golgi-associated membrane proteins such as
glycosyltransferases. They also suggested that attachment of the CMD to
the ER was essential for the Golgi localization of giantin.
The yeast Sed5p is a t-SNARE that is primarily localized to the Golgi
and involved in vesicular transport between the ER and the
Golgi complex (17). Syntaxin-2, which is also a t-SNARE, is,
however, localized to the plasma membrane (18). Both Sed5p and
syntaxin-2 are COOH-terminally anchored proteins with the same membrane
topology as giantin. The different localization of these proteins,
despite the same length of their CMDs, raises the possibility that the
Golgi localization signal of giantin and Sed5p is contained in a
cytoplasmic domain rather than in the CMD. In fact, it was reported
that Sed5p requires a cytoplasmic domain in addition to the CMD for
targeting to the Golgi (19). In the present study we carried out
mutational analysis of giantin to determine a domain responsible for
its targeting and retention to the Golgi, demonstrating that the
cytoplasmic domain of about 100 residues adjacent to the CMD is
essential for the localization of giantin to the Golgi complex.
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EXPERIMENTAL PROCEDURES |
Preparation of Species-specific Anti-giantin
Antibodies--
Giantin contains substantially different amino acid
sequences in several domains between rats and humans, which include
domains R1 (positions 547-617), R2 (1178-1239), and R3 (1668-1764)
for rat and H1 (positions 556-625), H2 (1188-1260), and H3
(1689-1784) for human. cDNA constructs encoding these domains were
fused with that of glutathione S-transferase. The
glutathione S-transferase fusion antigens were expressed in
and purified from bacteria, followed by immunization in rabbits as
described previously (20). Among the antibodies raised, anti-H1
antibody was found to be specific for giantin of humans and monkeys,
whereas anti-R2 antibody was specific for the rat antigen without
cross-reactivity to the human and monkey antigens, when examined by
immunofluorescence microscopy and immunoblotting.
Construction of Expression Plasmids--
The cDNA encoding
rat giantin/GCP364 (GW) (15) was inserted into the EcoRI
site of pSG5 expression vector. The cDNA encoding rat syntaxin-2
(S2W) was obtained by the method of reverse transcription-polymerase chain reaction using rat brain mRNA as a template and synthetic oligonucleotides 5'-ATGATATCATGCGGGACCGGCTGCCGGA-3' and
5'-TACTCGAGGAGGCAAGCACCCAGCGCCA-3' (18). The polymerase chain reaction
product was ligated downstream of the sequence encoding the Met-FLAGTM
tag for recognition by the monoclonal antibody M2 (21). A mutant of
giantin lacking the CMD (G
M) was generated by introducing the
termination codon TGA into the position Arg3163 (CGC). For
construction of the chimeric proteins G-SM (the giantin cytoplasmic
domain fused with the CMD of syntaxin-2) and S2-GM (the syntaxin
cytoplasmic domain with the giantin CMD), appropriate restriction
endonuclease sites were generated in the giantin cDNA (BbrPI at nucleotide number 9780) and in the syntaxin-2
cDNA (SmaI at nucleotide number 798) by site-directed
mutagenesis with synthetic oligonucleotides
5'-TTCCCGGACCCGCTGGATAATTGCTGCTGT-3' for giantin and
5'-AGACGGAAACCGTGGATAATTGCTG-3' and
5'-ACGGAAACCCGGGATAATTGCTGCT-3' for syntaxin-2. The mutated cDNAs
were digested with BbrPI or SmaI and reciprocally
fused with proper orientations. Amino acid residues at joining sites
were remutated for having the correct sequence. The COOH-terminally
deleted mutants D1-D5 were prepared by introducing the termination
codon into appropriate sites of the GW plasmid. The D5-SM chimera
was constructed by using G-SM as a template with
a synthetic oligonucleotide
(5'-CAGCTGCCTCCAGCCCAGCAACCCGCTGGATAATTGCTGC-3'). For
construction of NH2-terminally deleted mutants (D6-D9,
D6
M, D9
M, D9-SM, I-SM, II-SM, and III-SM), proper restriction
endonuclease sites protruding the blunt end were generated in
appropriate sites of GW, G
M, and G-SM, and the constructs were
digested with the proper restriction endonucleases. The mutants were
ligated in frame to the FLAG tag sequence. All the constructs prepared
by site-directed mutagenesis were verified by sequencing (21).
The cDNA encoding golgin-84 was obtained by the method of reverse
transcription-polymerase chain reaction from HeLa cell mRNA as a
template using synthetic oligonucleotides
5'-GAATTCATGTCTTGGTTTGTTGATCTTGC-3' and
5'-GAATTCATTTGCCATATGGTTGGTCGTGG-3' (22). The human syntaxin-5 cDNA was obtained by using synthetic oligonucleotides
5'-ATGGATCCATGTCCTGCCGGGATCGG-3' and 5'-ATGTTAACAGGCTCAGTGGCAGCACTGG-3'
(18). Truncated mutants of golgin-84 and syntaxin-5 were
constructed by introducing proper restriction endonuclease sites
protruding the blunt end with similar treatments as described above.
All the cDNA constructs were tagged with FLAG and inserted into the
expression vector pSG5.
Cell Culture and Transfection--
COS-1 cells and HeLa cells
were cultured as described previously (23). Each plasmid (10 µg) was
transfected into COS-1 cells with the Lipofectin reagent (21). At
20 h after transfection, cells were incubated at 37 °C with or
without cycloheximide (50 µg/ml) for 5 h and fixed for
immunofluorescence microscopy. For immunoblotting analysis, 20 µg of
each plasmid was transfected into cells using an electroporation
apparatus (21), and transfected cells were cultured for 20 h as above.
Immunofluorescence Microscopy and
Immunoblotting--
Immunofluorescence microscopy was carried out as
described previously (20). Primary antibodies used were as follows:
rabbit antiserum for human giantin (anti-H1) and for rat giantin
(anti-R2) (dilution factor for each, 1:50), mouse monoclonal anti-FLAG
M2 (1:100), and rabbit anti-
-COP IgG (15 µg/ml).
Rhodamine-conjugated goat anti-rabbit IgG (1:50) or fluorescent
isothiocyanate-conjugated anti-mouse IgG (1:50) was used as the
secondary antibody. For immunoblotting, proteins were separated by
SDS-PAGE (5% or 10% gels) followed by immunoblotting with the
indicated antibodies (at a 1:1000 dilution for each antibody). The
immunoreactive proteins were visualized using an ECL kit (21). In some
experiments the immunoblots were scanned with a GT-8500 scanner (Epson,
Inc., Tokyo, Japan) and analyzed by Adobe Photoshop (Adobe
Photosystems, Columbia, MD) and NIH Image software.
Preparation of a Golgi-enriched Fraction--
Transfected cells
were harvested with a cell scraper, washed twice with Dulbecco's
phosphate-buffered saline, and homogenized by 20 passages through a
25-gauge needle in 10 mM Tris-HCl (pH 7.5), 0.25 M sucrose, and a protease inhibitor mixture (21). The
homogenate was centrifuged at 600 × g for 5 min, and
the postnuclear supernatant (PNS) was separated into a membrane
fraction and a soluble fraction by centrifugation at 105,000 × g for 1 h. The Golgi fraction was prepared by the
method of Balch et al. (24). The PNS fraction of transfected
cells was mixed with PNS from HeLa S3 cells (~8 mg of protein),
adjusted to 1.4 M sucrose, and loaded on 0.8 M
and 1.2 M sucrose layers. After centrifugation at
73,000 × g for 2.5 h, a turbid band floated at
the 0.8 M/1.2 M sucrose interface was harvested
as the Golgi-enriched fraction. The Golgi fraction was found to contain
UDP-galactosyltransferase (55%), acid phosphatase (0.8%),
5'-nucleotidase (4.4%), calnexin (5.7%), and protein (2%), as
compared with the PNS fraction.
Other Analytical Methods--
Enzyme assays for
UDP-galactosyltransferase (25), acid phosphatase (26), and
5'-nucleotidase (27) were performed by the established methods.
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RESULTS |
Giantin Cytoplasmic Domain Is Sufficient for Golgi
Localization--
To examine the role of the cytoplasmic domain and
the CMD for targeting and localization of giantin to the Golgi
apparatus, we prepared cDNA constructs of rat giantin with or
without the CMD, syntaxin-2, and their chimeras (Fig.
1A), which were transfected into COS-1 cells. The expressed proteins were analyzed by
immunoblotting (Fig. 1B) and immunofluorescence microscopy
(Fig. 1C). The expressed proteins were detected by anti-R2
antibody (specific for rat giantin) or anti-FLAG. When cell homogenates
were separated into membranes and cytosol, all the expressed proteins
including giantin without CMD (G
M) were recovered in the membrane
fraction (Fig. 1B). Immunofluorescence microscopy revealed
that the mutant G
M was localized to the Golgi region, as observed
for the wild-type giantin GW (Fig. 1C).

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Fig. 1.
Expression of giantin with or without the
CMD. A, expression plasmids were constructed for
wild-type giantin (GW), giantin without the CMD (G M) or with the CMD
of syntaxin-2 (G-SM), wild-type syntaxin-2 (S2W), and syntaxin-2 with
the CMD of giantin (S2-GM). The position of FLAG is also shown.
B, homogenates prepared from COS-1 cells at 20 h after
transfection with the vector alone (Mock) or with the
indicated plasmids were fractionated into membrane (P) and
cytosol (S) fractions. The samples were analyzed by SDS-PAGE
(5% gels for giantin and 10% gels for syntaxin-2) and immunoblotting
with anti-R2 antibodies (lanes 1-8) or anti-FLAG antibodies
(lanes 9-12). C, at 20 h after transfection
with the indicated plasmids, cells were fixed and subjected to
immunofluorescence microscopy. The primary antibodies used were anti-R2
antibodies (GW, G M, and G-SM) or anti-FLAG monoclonal antibody (S2W
and S2-GM). Mock transfected cells stained for -COP are also
shown.
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Syntaxin-2 is also an integral membrane protein with the same membrane
topology as giantin but is localized to the plasma membrane (Fig.
1C, S2W). To confirm the role of the cytoplasmic domain of giantin for Golgi localization, we examined the intracellular distribution of chimeric proteins constructed by giantin and
syntaxin-2. A chimera of the giantin cytoplasmic domain with the CMD of
syntaxin-2 was localized to the Golgi region (Fig. 1C,
G-SM), whereas another chimera of the syntaxin-2 cytoplasmic
domain with the giantin CMD was expressed on the cell surface (Fig.
1C, S2-GM).
To reinforce these observations, we isolated a Golgi-enriched fraction
from the transfected cells and analyzed the expressed proteins by
immunoblotting (Fig. 2A). When
compared with PNS fractions, the giantin mutants without the CMD
or substituted with the syntaxin-2 CMD were recovered in the
Golgi-enriched fraction at the same level as the wild-type giantin
(Fig. 2B). These were in contrast with the recovery of the
wild-type syntaxin-2, although the syntaxin-2 mutant replaced by the
giantin CMD was significantly recovered in the Golgi fraction.

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Fig. 2.
Recovery of expressed proteins in the Golgi
fraction. A, PNS fractions were prepared from cells
transfected with the indicated plasmids and used for isolation of the
Golgi-enriched fraction. Both the PNS and Golgi fractions were analyzed
by SDS-PAGE and immunoblotting, as shown in Fig. 1. B, the
recovery of each protein in the Golgi fraction was quantified from the
data shown in A. The values are expressed as the means ± S.D. (n = 4) by taking those in the PNS as 100%.
C, experiments were carried out to rule out the possibility
that the endogenous giantin is involved in the dimer formation with the
exogenous proteins. Lysates prepared from cells transfected with the
indicated plasmids were used for immunoprecipitation with anti-R2
(lanes 2-4 and 6-8) or anti-H1 (lanes
1 and 5). The immunoprecipitate was analyzed by
SDS-PAGE and immunoblotting with anti-R2 (lanes 1-4) or
anti-H1 (lanes 5-8).
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It has been suggested that giantin forms a disulfide-linked homodimeric
conformation in vivo and associates with other membrane proteins or cytoskeletal proteins (12, 16). This raises the possibility
that giantin without the CMD expressed in COS-1 cells may form dimers
with the endogenous one and be cotransported to the Golgi apparatus. To
test this possibility, cell lysates were subjected to
immunoprecipitation with anti-R2 antibody (specific for the exogenously
introduced giantin), and the resultant immunoprecipitates were analyzed
by SDS-PAGE and immunoblotting with anti-R2 or anti-H1 (specific for
the endogenous giantin). When blotting was probed with anti-R2, the
mutants G
M and G-SM, as well as the wild-type GW, were detected in
the transfected cells (Fig. 2C, lanes 2-4). In
contrast, when probed with anti-H1, no endogenous giantin was detected
(Fig. 2C, lanes 6-8). The endogenous giantin was
detected with anti-H1 only when it was used for both
immunoprecipitation and immunoblotting (Fig. 2C, lane
5). Thus, it is unlikely that the exogenously introduced giantin
molecules are localized to the Golgi by association with the endogenous
giantin. Taken together, these results suggest that the cytoplasmic
domain of giantin itself has a signal for its targeting and
localization to the Golgi apparatus.
Essential Domain of Giantin Required for Golgi
Localization--
The cytoplasmic domain of giantin required for Golgi
localization was examined by deletion analysis, for which a series of COOH-terminally truncated mutants (D1-D5) were constructed and expressed in COS-1 cells (Fig.
3A). When cell homogenates
were separated into membranes and cytosol, the smallest
NH2-terminal fragment, D1, was recovered exclusively
in the cytosolic fraction (Fig. 3B, lanes 1 and
2). The amount of the mutant proteins associated with
membranes was increased as they contained longer extensions to the COOH
terminus (Fig. 3B, lanes 3-8). The longest
mutant, D5, lacking a small COOH-terminal segment, was completely
recovered in the membrane fraction, as observed for D5-SM with the
syntaxin-2 CMD (Fig. 3B, lanes 9-12). When cells
were examined by immunofluorescence microscopy, three mutants (D1-D3)
were detected throughout the whole cell including the cell periphery,
whereas the membrane-bound forms D4 and D5 showed a profile lacking the
cell periphery staining, clearly different from that of the above three
mutants, D1-D3 (Fig. 3C). D4 and D5 appeared to be
localized mostly to the ER, because the two mutants were colocalized
with the ER marker calnexin (data not shown). To exclude the
possibility that no localization of D5 to the Golgi is due to the lack
of CMD, we examined the cellular distribution of D5-SM, which contained
the CMD of syntaxin-2 at the COOH terminus (Fig. 3A,
D5-SM), demonstrating that the mutant also showed a typical
ER staining pattern (Fig. 3C). These results indicate that
the Golgi targeting signal is present in the COOH-terminal segment
beyond the amino acid residue 2803. This was confirmed by analysis of
another set of NH2-terminally truncated mutants with or
without the CMD (Fig. 4A). All
the expressed proteins were found to be recovered in the membrane
fraction (Fig. 4B) and localized to the Golgi region (Fig.
4C), irrespective of the presence or absence of the CMD.

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Fig. 3.
Intracellular distribution of COOH-terminally
deleted mutants. A, shown are giantin mutants with
various sizes of COOH-terminal deletion (D1-D5) and D5 with the
syntaxin CMD (D5-SM). D1, D2, and D3 were designed to contain one, two,
and three major coiled-coil domains, respectively, of the four
(open boxes in the top schema). B,
cells expressing the indicated mutants were fractionated into membrane
(P) and cytosol (S) fractions. The samples were
analyzed by SDS-PAGE and immunoblotting with antibodies to R1, R2, and
R3. C, cells expressing the indicated mutants were observed
by immunofluorescence microscopy, for which anti-R1, -R2, and
-R3 antibodies were used as the primary antibodies.
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Fig. 4.
Intracellular distribution of
NH2-terminally deleted mutants. A,
schematic representation of giantin mutants with various sizes of
NH2-terminal deletion (D6-D8) and additional deletion of
the CMD (D6 M-D8 M). Each mutant was tagged with the FLAG at the
NH2 terminus. The expressed giantin mutants were analyzed
by immunoblotting (B) and immunofluorescence microscopy
(C) as described in the legend to Fig. 1, using anti-FLAG
antibody, instead of anti-R2.
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The COOH-terminal domain was further characterized for Golgi targeting
and localization, for which three mutants, D9 (2804-3187 amino acid
segment of giantin), D9
M (D9 without CMD), and D9-SM (substituted
with syntaxin-2 CMD), were constructed (Fig.
5A). Both the expressed D9 and
D9-SM were detected in the Golgi region, colocalizing with the
endogenous giantin (Fig. 5B). The mutant D9
M without the
CMD, however, showed a diffuse distribution throughout the cell. This
is in contrast with the clear Golgi localization of the longer segment
D8
M (Fig. 4C). Possible reasons for the different
behavior of D8
M and D9
M will be discussed later.

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Fig. 5.
Requirement of the CMD for Golgi targeting of
D9 with a COOH-terminal domain. A, mutants of a
COOH-terminal domain with (D9) or without the CMD (D9 M) or with the
CMD of syntaxin-2 (D9-SM) were constructed and tagged with the FLAG at
the NH2 terminus. B, the mutants expressed were
immunostained with anti-FLAG (upper panels), as shown in
Fig. 1. The endogenous giantin was coimmunostained with anti-H1
(B, lower panels).
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As shown in Figs. 3C and 4B, the expressed mutant
proteins were localized to the compact spot-like structure in the
juxtanuclear region, which is a characteristic feature of the Golgi
complex found in COS-1 cells (15, 28). Recently, however, it has been shown that a similar compact structure, the aggresome, is also formed
in a juxtanuclear region by misfolded proteins (29, 30). To distinguish
true Golgi localization from the formation of aggregates, we carried
out a more careful colocalization analysis with endogenous giantin
in HeLa cells, where the Golgi structure is less compact than that in
COS-1 cells. Both the mutants D8
M and D9-SM expressed were
colocalized well with endogenous giantin in perinuclear regions corresponding to the Golgi (Fig. 6,
A and B, a and b). When the cells were incubated with brefeldin A for 30 min, the mutants, as well
as endogenous giantin, were dispersed into the cytoplasm (Fig. 6,
A and B, c and d). Further
incubation of the cells without the drug allowed each protein to
relocalize to the perinuclear regions (Fig. 6, A and
B, e and f). Taken together, these
results support the conclusion that the expressed mutants are localized to the Golgi, not clustered into the aggresome.

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Fig. 6.
The effect of brefeldin A on the Golgi
localization of giantin and mutants. HeLa cells expressing D8 M
(A) or D9-SM (B) were incubated in the presence
of brefeldin A (2 µg/ml) for 30 min (c-f) and
further incubated for 90 min after the drug was removed from the medium
(e and f). The cells, including those before the
drug treatment (a and b), were fixed and
double-immunostained with anti-FLAG for each mutant (a,
c, and e) and with anti-H1 for the endogenous
wild-type giantin (b, d, and f)
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The COOH-terminal D9 sequence is predicted to contain three coiled-coil
domains. We constructed shorter segments that contained one of the
coiled-coil domains (I, II, and III) and were supplemented with the
syntaxin-2 CMD (SM) (Fig. 7A).
When these were expressed, only the construct containing the domain III
was found to localize to the Golgi (Fig. 7B,
III-SM). In addition, the insertion of the domain III into
syntaxin-2 prevented transport of syntaxin-2 to the plasma membrane,
resulting in its retention in the Golgi (Fig. 7B,
S2/III). These results suggest that the domain III with the
coiled-coil domain adjacent to the CMD is essential for Golgi localization of giantin. Shorter fragments of III-SM failed to be
targeted to the Golgi (data not shown).

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Fig. 7.
The essential domain for Golgi targeting and
retention. A, coiled-coil probability of the mutant
D9-SM and schematic illustration of constructs containing each
coiled-coil domain (I, II, and III) with the syntaxin-2 CMD (SM). A
mutant of syntaxin-2 containing the giantin domain III (S2/III) is also
shown. Each mutant was tagged with the FLAG at the NH2
terminus. B, the mutants expressed were analyzed by
immunofluorescence microscopy with anti-FLAG.
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Common Features of COOH-terminally Anchored Golgi
Proteins--
Syntaxin-5 is a mammalian homologue of Sed5p localized
to the Golgi (19, 31), which has the same membrane topology as giantin
and contains a coiled-coil domain adjacent to the CMD (Fig.
8A). Syntaxin-5-derived
constructs similar to those of giantin were prepared and expressed in
COS-1 cells. When observed by immunofluorescence microscopy (Fig.
8B), syntaxin-5 without the CMD (S5
M) or substituted with
syntaxin-2 CMD (S5-SM) was localized to the Golgi, as observed for
similar constructs of giantin. However, syntaxin-5 lacking an
additional COOH-terminal cytoplasmic domain (S5D1) was detected throughout the cytoplasm. In contrast, the COOH-terminal cytoplasmic fragment of 79 residues, without the CMD (S5D2) or supplemented with
syntaxin-2 CMD (S5D2-SM), was localized to the Golgi.

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Fig. 8.
The essential domain of syntaxin-5 for Golgi
localization. A, coiled-coil probability of syntaxin-5
and schematic representation of wild-type (S5W) and mutant constructs
of syntaxin-5: S5 M lacking the CMD (closed box), S5-SM
containing the CMD of syntaxin-2 (shaded box), and deletion
mutants without (S5D1 and S5D2) or with the CMD of syntaxin-2
(S5D2-SM). Each construct was tagged with the FLAG at the
NH2 terminus. B, the wild type and mutants of
syntaxin-5 expressed in COS-1 cells were observed by immunofluorescence
microscopy after being stained with anti-FLAG.
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Golgin-84 is a transmembrane protein consisting of a long cytoplasmic
coiled-coil domain, a TMD, and a short luminal domain of 16 residues
(22) (Fig. 9). Essentially the same
results as those obtained for giantin and syntaxin-5 were obtained for
golgin-84 by mutational analysis. Golgin-84 without the TMD and luminal domain (g84
T) or substituted with the syntaxin-2 CMD (g84-SM) was
localized to the Golgi. However, golgin-84 without the COOH-terminal 161 residues (g84D1) was detected throughout the cytoplasm. It was
confirmed that the COOH-terminal cytoplasmic domain of 125 residues was
required for the Golgi localization of golgin-84 (Fig. 9B).
This domain contains a part of the coiled-coil domains. Additional
deletion of the coiled-coil domain (positions 569-623) resulted in
distribution of the mutant throughout the cytoplasm (data not shown).

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Fig. 9.
The essential domain of golgin-84 for Golgi
localization. A, coiled-coil probability of golgin-84
and schematic representation of wild-type (g84W) and mutant constructs
of golgin-84; the closed box indicates the TMD of golgin-84,
and shaded boxes indicate the CMD of syntaxin-2. Each
construct was tagged with the FLAG at the NH2 terminus.
B, the proteins expressed in COS-1 cells were observed by
immunofluorescence microscopy after being stained with anti-FLAG.
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DISCUSSION |
Giantin is an integral membrane protein of which the single
hydrophobic domain CMD at the COOH terminus functions as the anchor to
the Golgi membrane (12-15). It was proposed that the localization of
giantin to the Golgi involves at least three steps: insertion into the
ER membrane, controlled incorporation into transport vesicles, and
retention within the Golgi (16). The CMD of giantin was suggested to be
essential for the insertion into the ER membrane and transport to the
Golgi, because deletion of the CMD caused no attachment of giantin to
the ER, resulting in its distribution into the cytoplasm (16). This is
in contrast to our results, which demonstrated that giantin lacking the
CMD is still associated with the ER and transported to the Golgi,
although we agree with the proposal that newly synthesized giantin is
initially associated with the ER and then transported to the Golgi,
like conventional membrane proteins of the secretory pathway (16).
Giantin appears to have another cytoplasmic domain, in addition to the
CMD, for the initial association with the ER membrane, because the
mutant D5 without a COOH-terminal domain after position 2804 was
completely associated with the ER membrane (Fig. 3). Although the
mutant D5 was not transported to the Golgi, the mutant D8
M
(2619-3162) was localized to the Golgi (Fig. 4). In addition, the
mutant D9
M (2804-3162) was neither associated with the ER nor
transported to the Golgi, whereas the counterpart D9 with the CMD was
localized to the Golgi (Fig. 5). Taken together, these results suggest
that giantin contains domains with the following characteristics. 1) A cytoplasmic domain between positions 2619 and 2804 interacts with the ER membrane but is not sufficient to target
the protein to the Golgi. 2) The Golgi targeting domain is contained in
the sequence 2804-3162. 3) The lack of the cytoplasmic domain
(2619-2804) for the ER interaction can be compensated by the CMD, and
the presence of either domain of the two is prerequisite for the ER
association and subsequent transport to the Golgi. It is also clear
that the CMD of giantin does not function as the primary determinant
for Golgi localization, as supported by the finding that its deletion
or substitution with the CMD of syntaxin-2 causes no effect on the
Golgi localization of giantin. This is in contrast to other
Golgi-resident type II and III membrane proteins of which the TMD is
involved in their Golgi localization (5, 6).
Exit from the ER is now considered to be a selective event involving
sorting and concentration, in contrast to a constitutive "bulk
flow" process as suggested previously (32). Recent studies have shown
that a diacidic sorting signal is required for efficient ER export (33,
34). This motif, which contains the amino acids Asp or Glu separated by
a variable residue ((D/E)X(E/D)), is found in the
cytoplasmic tail of a number of transmembrane proteins and is suggested
to facilitate interaction with the COPII coat machinery. The
di-acidic exit motif is located within or close to the Tyr-based
sorting motif YXXØ (where Ø is a bulky hydrophobic residue). Thus, it is likely that the ER accumulation of giantin mutants observed here is due to the lack of the ER exit motif. In fact,
the motif is not found in the sequence of mutant D5 (1-2803), whereas
it is found in positions 2898-2906 (YLMAISDKD)
and in two other positions, 3053-3055 (ETE) and 3086-3088 (EEE), all or one of which is contained in the competent mutants D6-D9 and I-III with or without the CMD.
It has been suggested that the TMD of Golgi-resident type II and III
membrane proteins is involved in Golgi retention or localization; the
TMD-dependent localization probably reflects the lipid
composition and thickness of target membranes (5, 6, 8). Although not
containing any obvious distinguishing sequence motif, the Golgi TMDs
are on average five residues shorter than those of plasma membrane
proteins. The relevance of this difference in TMD length to retention
is supported by mutational analysis; lengthening the TMD of Golgi
enzymes results in their transport to the plasma membrane (5-7). It
is, however, unlikely that the TMD or CMD of giantin is critically
involved in Golgi localization. Giantin has the same residue number in
the CMD as that of the plasma membrane protein syntaxin-2, and
replacement of the CMD by that of syntaxin-2, or even the lack of the
CMD, does not influence the Golgi localization of giantin.
In this study we also examined two other COOH-terminally anchored
proteins, golgin-84 and the t-SNARE syntaxin-5, which are localized to
the Golgi with the same membrane topology as giantin. Because giantin
has been shown to tether COPI vesicles to the Golgi membrane
(35), golgin-84 was suggested to play a similar role in tethering
transport vesicles to a target membrane (22). Our results demonstrated
that the three COOH-terminally anchored proteins have the common
feature for Golgi localization; each cytoplasmic domain of ~100 amino
acid residues close to the CMD or TMD is primarily required for the
targeting and retention to the Golgi. This is essentially the same
result obtained for the yeast Sed5p (17). The cytoplasmic domain of
syntaxin-5 required for the Golgi localization is highly conserved in
that of Sed5p (36). The importance of a similar cytoplasmic domain for
the Golgi retention was also demonstrated for the Golgi enzyme Mnt1p (37) and the trans-Golgi network-localized t-SNARE
syntaxin-6 (38). Syntaxin-6 has a cytoplasmic H2 domain adjacent to the TMD, which is predicted to form a 63-residue amphipathic
-helical structure and play a major role in Golgi localization (38). In
contrast, the plasma membrane syntaxin-1 was suggested to have no
signal for the ER attachment or the Golgi localization in the cytoplasmic domain (39).
In addition to giantin and golgin-84, there are many other coiled-coil
proteins that are peripherally associated with the cytoplasmic face of
the Golgi, including GM130/golgin-95 (40), golgin-97 (41),
golgin-160/GCP170 (20, 42), and golgin-245/p230 (42, 43). Golgin-97,
golgin-245/p230, and several other proteins are found to contain a
conserved COOH-terminal sequence (about 50 amino acids), which is
designated the GRIP domain (44), Rab6-interacting domain (45),
or Golgi localization domain (46). The domain contains a
consensus sequence including a Tyr-based motif. In fact, the deletion
of the domain or substitution of Tyr by Ala in the Tyr-based motif
resulted in failure of the proteins to correctly localize to the
Golgi (44-46), suggesting that this domain functions in targeting
these coiled-coil proteins to the Golgi. It is of interest to know
whether giantin also contains such a motif in the corresponding domain.
The consensus sequence proposed for the GRIP domain, however, is
not found in the COOH-terminal cytoplasmic domain (domain III) of
giantin or in that of golgin-84 (g84D2) or syntaxin-5 (S5D2). At
present we cannot find a possible consensus sequence or motif among the
domains of giantin, golgin-84, and syntaxin-5, except for the common
structural feature that they contain a coiled-coil domain. This
suggests the possibility that the COOH-terminal coiled-coil domain may
play an important role in the interaction of giantin (as well as
golgin-84 and syntaxin-5) with the Golgi membrane, although
details of the mechanism remain to be solved.