School of Life Science, Tokyo University of Pharmacy and Life Science,
Hachioji, Tokyo 192-0392, Japan
* Present address: Department of Life Science, Graduate School and Faculty of
Science, Himeji Institute of Technology, Ako-gun, Hyogo 678-1297, Japan
Author for correspondence (e-mail:
tagaya{at}ls.toyaku.ac.jp)
Accepted 4 August 2002
![]() |
Summary |
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Key words: Golgi apparatus, Heterotrimeric G protein, Regulator of G protein signaling
![]() |
Introduction |
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Recently, the functions of heterotrimeric G proteins in various
endomembrane systems have also been recognized
(Lang, 1999). These include
protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus
(Schwaninger et al., 1992
),
intra-Golgi transport (Helms et al.,
1998
), cargo sorting and vesicle budding from the
trans-Golgi network (Leyte et
al., 1992
; Pimplikar and
Simons, 1993
), control of exocytosis
(Ohara-Imaizumi et al., 1992
;
Aridor et al., 1993
;
Vitale et al., 1993
;
Ohnishi et al., 1997
), and
autophagic sequestration (Ogier-Denis et
al., 1996
).
In mammalian cells the Golgi apparatus is of fundamental importance for the
secretion and posttranslational modification of secretory and membrane
proteins. The Golgi apparatus consists of a set of highly dynamic membrane
compartments that are maintained through the continuous anterograde and
retrograde flow of proteins and lipids. The Golgi apparatus undergoes
disassembly and reassembly during mitosis, and there is a cell cycle-dependent
mechanism that controls the Golgi structure and thus ensures the fidelity and
reliability on the partitioning of this organelle into daughter cells
(Warren and Malhotra, 1998).
Golgi disassembly at the onset of mitosis may be controlled by signaling
proteins at the periphery of Golgi membranes
(Nelson, 2000
).
We and others have previously shown the involvement of heterotrimeric G
proteins in regulation of the Golgi structure
(Hidalgo et al., 1995;
Jamora et al., 1997
;
Jamora et al., 1999
;
Yamaguchi et al., 1997
;
Yamaguchi et al., 2000
).
However, it is currently unclear as to which subunit of heterotrimeric G
proteins is involved in this regulation, and little is known about the
mechanism by which G proteins exert their actions. A study involving a
semi-intact cell system demonstrated that Gß
causes vesiculation
of the Golgi apparatus through direct activation of protein kinase D
(Jamora et al., 1999
). On the
other hand, we showed that the activation of G proteins by GTP
S or
AlF4- protects the Golgi apparatus from disassembly
caused by nordihydroguaiaretic acid (NDGA). Consistent with the involvement of
G
-mediated signaling in Golgi organization, the addition of
Gß
can reverse the effect of GTP
S on NDGA-induced Golgi
disassembly through reformation of the heterotrimer
(Yamaguchi et al., 1997
).
Furthermore, overexpression of G
i2 or G
z,
both of which are
i family members, results in attenuation
of the NDGA effect (Yamaguchi et al.,
2000
). Although our data comprise circumstantial evidence that
G
i2 and/or G
2 are involved in Golgi
organization, it is not clear whether these G proteins actually regulate the
Golgi structure in normal living cells.
Recently, a novel family of G protein regulators, i.e. regulators of G
protein signaling (RGS) proteins, has emerged
(Berman and Gilman, 1998;
Hepler, 1999
;
De Vries et al., 2000
). RGS
proteins stimulate the intrinsic GTPase activity of heterotrimeric G proteins,
thereby decreasing the concentration of active G
in cells. In addition
to this effect, RGS proteins prevent the binding of G
to their
effectors. Therefore, RGS proteins serve as negative regulators of G
protein-mediated signaling pathways (Berman
and Gilman, 1998
; De Vries et
al., 2000
).
RGS proteins appear to be ideal tools for demonstrating the physiological
relevance of our finding that active G subunits prevent NDGA-induced
Golgi disassembly. In this study, we overexpressed various RGS proteins that
preferentially inactivate a different set of G proteins, and examined the
Golgi morphology. The results suggest that, indeed, active
G
z, but not G
i2, is necessary for
maintenance of the integrity of the Golgi structure.
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Materials and Methods |
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Plasmids
The cDNAs for RGS4, GAIP, RGSZ1 and RGSZ2 were kindly donated by Drs.
Elliott Ross and Tohru Kozasa (University of Texas Southwestern Medical
Center), and subcloned into mammalian expression vectors pcDNA3 (Invitrogen)
and pFLAG-CMV-6 (Sigma). The full-length cDNA encoding human RGS2 was
amplified from a human leukocyte cDNA library (Clontech) by means of the
polymerase chain reaction (PCR) with primers corresponding to the initiation
and termination sites of RGS2. To obtain the full-length cDNA encoding RGS3,
nucleotides corresponding to the N-terminal and C-terminal regions were
independently amplified from the human cDNA library by PCR, and then cloned
into pFLAG-CMV-6. The expression plasmid for the vesicular stomatitis virus
ts045 G protein fused to the green fluorescent protein (VSVG-GFP) was a
generous gift from Dr Jennifer Lippincott-Schwartz (National Institutes of
Health, USA).
Site-directed mutagenesis
A rat cDNA containing the entire coding sequence of Gz
was cloned into a eukaryotic expression vector, pALTER-MAX (Promega).
G
z(G204A/E246A/A327S), in which Gly-204, Glu246, and Ala-327
of G
z were replaced with Ala, Ala, and Ser, respectively,
was generated by oligonucleotide-directed mutagenesis using an Altered Sites
II Mammalian Mutagenesis System (Promega). A G
binding deficient mutant
of RGSZ1, RGSZ1(E116A/N117A), in which Glu-116 and Asn-117 were individually
substituted by Ala, was prepared by PCR-mediated mutagenesis.
Cell culture and transient transfection
HeLa, BHK, and Clone9 cells were cultured at 37°C in -minimum
essential medium supplemented with 10% fetal bovine serum, 50 IU/ml
penicillin, and 50 µg/ml streptomycin, under humidified air containing 5%
CO2. PC12 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, 10%
fetal bovine serum, and 5% horse serum. Cells were transfected by lipofection
using LipofectAMINE PLUS according to the manufacturer's instructions (Life
Technologies).
Indirect immunofluorescence analysis
Indirect immunofluorescence microscopy was performed essentially as
described previously (Tagaya et al.,
1996). Cells were grown on 15-mm diameter glass coverslips in
12-well tissue culture plates. At the indicated times after transfection,
cells were subjected to fixation in 4% paraformaldehyde in phosphate-buffered
saline (PBS) for 20 minutes at room temperature, followed by permeabilization
with 0.2% Triton X-100 in PBS for 20 minutes. They were then incubated for 30
minutes in PBS containing 2% bovine serum albumin (BSA), and incubated for 1
hour at 37°C with appropriate primary antibodies in PBS-2% BSA. The cells
were washed three times with PBS, and then stained with FITC- and/or Texas
red-conjugated secondary antibodies. An Olympus BX50 fluorescence microscope
was used for routine immunofluorescence analysis. Confocal images were
obtained with an Olympus Fluoview 300 laser confocal microscope. For the
transferrin internalization assay, transfected cells were incubated in the
presence of 25 µg/ml of FITC-conjugated transferrin (Sigma) for 1 hour at
37°C. After extensive washing in PBS, they were fixed with 4%
paraformaldehyde in PBS. Since the fluorescence intensity of FITC-conjugated
transferrin was weak, the fixed cells were further stained with the
anti-transferrin rabbit polyclonal antibody and the FITC-conjugated secondary
antibody.
Semi-quantitative reverse transcription-PCR (RT-PCR)
Total RNA was isolated from cells using isogen reagent (Nippon Gene) and
then used as the template for RT-PCR analysis. RT-PCR reactions were performed
with a BcaBEST RNA PCR kit (Takara). PCR was performed for 45 and 20 cycles
for Gz and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
respectively, using the following sense and antisense primers:
G
z, 5'-CACCTGGAGGACAACGCCGCT-3' and
5'-TTTCGGTTTAGGTCCTCGAACTGA-3' (500-bp product); GAPDH,
5'-CATGGAGAAGGCTGGGGCTC-3' and
5'-CTCAGTGTAGCCCAGGATGC-3' (523-bp product), respectively. The
number of cycles was pre-determined to fall within the linear range of
amplification of each PCR product. The PCR products were electrophoresed on
1.6% agarose gels, stained with ethidium bromide, and visualized by UV
irradiation.
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Results |
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RGS proteins can attenuate G-protein mediated signaling pathways by acting
as GAPs for the Gi, G
q, and
G
12/13 families, but not the G
s one. To
date, more than 20 RGS proteins in mammalian tissues have been identified.
Certain RGS proteins exhibit high selectivity for a single or a few G
species (Berman and Gilman,
1998
; De Vries et al.,
2000
). RGSZ1 (Glick et al.,
1998
; Wang et al.,
1998
) and RGSZ2 show high selectivity for
G
z.
By immunofluorescence microscopy, we first examined the localization of a
Golgi marker ß-COP in HeLa cells expressing N-terminal
hexahistidine-tagged (6xHis-) RGSZ1. Although nontransfected cells
showed a typical ribbon-like Golgi structure, a dispersed or punctate
ß-COP staining pattern was frequently observed in cells overexpressing
6xHis-RGSZ1 (Fig. 1). The
degree of ß-COP dispersal was dependent on the level of RGSZ1 expression.
When RGSZ1 was expressed at high levels, ß-COP was almost completely
dispersed (Fig. 1a). On the
other hand, expression of RGSZ1 at moderate levels yielded a punctate or large
dot-like ß-COP staining pattern (Fig.
1c), which might represent fragmented Golgi. Approximately 50% of
the cells expressing a detectable level of RGSZ1 showed a completely or
partially disrupted ß-COP staining pattern, whereas less than 7% of the
nontransfected ones did. The latter value may represent the percentage of
cells in the late G2 and M phases. It is known that the Golgi
ribbon-like structure is reorganized into a punctate structure and exhibits a
more perinuclear localization at the G2/M transition, and then is
completely dispersed (Shima et al.,
1998) or transported back to the ER
(Zaal et al., 1999
) during
mitosis. The effect of the RGS proteins on the Golgi structure is not peculiar
to HeLa cells, similar results being obtained for BHK and Clone9 cells (data
not shown).
|
Selective attenuation of the Gz function induces
Golgi disassembly
We next examined whether or not other RGS proteins exert similar effects on
the Golgi apparatus. RGSZ2 is another Gz-specific RGS that
exhibits 62.7% amino acid identity with RGSZ1. GAIP, which is most similar to
RGSZ1 (62.6% amino acid identity) and RGSZ2 (57.6% amino acid identity),
exhibits GAP activity toward all G
i family members
(De Vries et al., 1995
;
Berman et al., 1996
). RGS2
exhibits high selectivity for G
q
(Heximer et al., 1997
). RGS3
is highly selective for the G
i family except for
G
z (Scheschonka et al.,
2000
). RGS4 stimulates the GTPase activity of both
G
i and G
q
(Berman et al., 1996
;
Watson et al., 1996
). These
RGS proteins were expressed as FLAG-tagged proteins in HeLa cells, and then
the Golgi morphology was examined. As shown in
(Fig. 2A, expression of RGSZ2
as well as that of RGSZ1 induced the dispersal of ß-COP, whereas
expression of GAIP, RGS2, RGS3, or RGS4 had essentially no effects on
ß-COP staining. The number of cells expressing a detectable level of RGS
proteins was determined, and the percentage of cells with a disrupted
ß-COP staining pattern was determined
(Fig. 2B). More than 30% of the
FLAG-RGSZ1- or FLAG-RGSZ2- expressing cells showed a dispersed ß-COP
staining pattern, whereas no significant dispersal of ß-COP was observed
for other RGS-expressing cells compared to control cells. These results
suggest that the dispersal of ß-COP by RGSZ1 and RGSZ2 is specific.
|
RGSZ1 stimulates Golgi disruption without markedly affecting other
cellular structures or functions
Since ß-COP is a peripheral membrane protein that undergoes
association and disassociation with the Golgi apparatus in an ARF1-dependent
manner (Donaldson et al., 1992;
Palmer et al., 1993
), the
results shown in Figs 1 and
2 may imply that the expression
of RGSZ1 merely induces the release of ß-COP from Golgi membranes. To
test this possibility, we examined the localization of several
Golgi-associated peripheral and integral membrane proteins in RGSZ1-expressing
cells. As shown in Fig. 3a-d,
peripheral Golgi proteins GM130 and p115 showed scattered staining patterns
and were distributed as distinct dots around the nucleus in RGSZ1-expressing
cells. As shown in Fig. 3e-h,
cis-Golgi integral membrane proteins membrin and syntaxin 5 exhibited similar
fragmented patterns in RGSZ1-expressing cells. Man II, a Golgi-resident
integral membrane protein, also showed a scattered distribution
(Fig. 3i-l). These results
demonstrate that RGSZ1 does not merely induce the release of peripheral
proteins from the Golgi membrane, but also causes the fragmentation of entire
Golgi stacks.
|
We wondered whether or not the fragmentation of the Golgi apparatus is a
consequence of the disassembly of microtubules, which are known to play an
important role in maintaining the Golgi structure
(Lippincott-Schwartz, 1998;
Thyberg and Moskalewski,
1999
). As shown in Fig.
4a,b, expression of RGSZ1 had no effect on the structure of
microtubules. In addition, expression of RGSZ1 did not conspicuously affect
the ER structure in most cells (Fig.
4c,d), although the formation of punctate aggregations was
observed in some cells (data not shown). RGSZ1 expression also did not affect
the uptake of FITC-transferrin (Fig.
4e,f). These results demonstrate that overexpression of RGSZ1
affects the Golgi structure without marked effects on other cellular
structures or functions.
|
Effect of RGSZ1 expression on vesicular transport
We next examined whether or not RGSZ1 expression perturbs anterograde
vesicular transport. For this purpose, a temperature-sensitive VSVG mutant
protein fused to GFP (VSVG-GFP) (Presley
et al., 1997) was used. HeLa cells were co-transfected with
plasmids for VSVG-GFP and FLAG-RGSZ1 and incubated at 40°C for 20 hours,
and then the temperature was changed to 32°C to initiate transport from
the ER. At 1 hour after the temperature shift, VSVG-GFP had moved from the ER
to the plasma membrane through the Golgi in 80% of the control cells
(Fig. 5). In RGSZ1-expressing
cells, the transport of VSVG-GFP was significantly delayed. At 1 hour after
the temperature shift, VSVG-GFP had reached the plasma membrane in 40% of the
RGSZ1-expressing cells. In the other RGSZ1-expressing cells, VSVG-GFP was
detected in dot-like structures that might represent fragmented Golgi
membranes. Essentially the same results were obtained for BHK and Vero
cells.
|
The inhibitory effect of RGSZ1 on the transport of VSVG-GFP might be underestimated. We noticed that the extent of Golgi dispersion was lower in cells expressing both RGSZ1 and VSVG-GFP than in those expressing RGSZ1 alone. In many cells, fragmented, large dot-like Golgi structures rather than completely dispersed ones were observed. It seemed likely that VSVG-GFP was transported to the plasma membrane through the perinuclear dot-like structures marked by Man II (Fig. 6a-c), ß-COP (Fig. 6d-f), and ERGIC-53 (Fig. 6g-i). We speculate that the transport of VSVG-GFP would be severely inhibited if the Golgi apparatus were completely dispersed by high expression of RGSZ1.
|
Association of RGSZ1 with Gz is required for
RGSZ1-mediated Golgi dispersal
The RGS family is defined by the RGS-box that binds to G subunits
and is responsible for the GAP function. A point mutation in the RGS-box of
RGS4 abrogates its ability to bind to G
and therefore is not able to
inactivate G
(Druey and Kehrl,
1997
; Srinivasa et al.,
1998
). To determine whether or not the GAP activity of RGSZ1 is
required for Golgi dispersion, RGSZ1(E116A/N117A), in which Glu-116 and
Asn-117 substituted individually by Ala, was constructed and assessed as to
its Golgi disassembling activity. This mutation was designed in analogy to the
RGS4 mutation (Srinivasa et al.,
1998
). A similar mutation has been successfully used for
assessment of the function of RGS3
(Scheschonka et al.,
2000
).
We first tested whether RGSZ1(E116A/N117A), as expected, cannot bind to
Gz. The wild-type or mutated FLAG-RGSZ1 was co-expressed in
cells with an active mutant of G
z, G
z(QL),
in which Gln-205 was replaced with Leu
(Fig. 7A). The wild-type RGSZ1,
which mainly remains in the cytosol (Fig.
3), was efficiently recruited to the plasma and internal
membranes, and co-localized with expressed G
z(QL),
indicating the interaction of RGSZ1 with G
z in cells. In
contrast, the mutated RGSZ1 remained in the cytosol and was not colocalized
with expressed G
z(QL). This indicates that the mutated RGSZ1
we constructed has the expected functional property in cells.
|
As shown in Fig. 7B, cells expressing wild-type RGSZ1 showed a dispersed ß-COP staining pattern (Fig. 7Ba,b). In contrast, RGSZ1(E116A/N117A) had no detectable effect on the distribution of ß-COP (Fig. 7Bc,d). The level of expression of the RGSZ1 mutant was comparable to that of the wild-type one (Fig. 7C). Similar results were obtained when Clone9 cells were used (data not shown).
Expression of a dominant-negative Gz subunit causes
Golgi disruption
As an alternative approach for investigating the effect of attenuation of
the Gz function, we employed a dominant-negative mutant
strategy. Several mutants possessing mutations in the conserved
nucleotide-binding region of GTPases including the
-subunits of
heterotrimeric G proteins have been characterized. The mutant we constructed
was a triple one (G204A/E246A/A327S), in which Gly-204, Glu-246, and Ala-327
were replaced with Ala, Ala, and Ser, respectively. This mutant is equivalent
to a triple mutant of G
s, which has decreased guanine
nucleotide-binding ability, and dominantly inhibits receptor-mediated hormonal
activation of G
s by sequestering Gß
and
activated receptors (Iiri et al.,
1999
).
The triple Gz mutant was not co-localized with RGSZ1 when
co-expressed, suggesting that the G
z mutant is not in a
GTP-bound active state in cells (data not shown). The triple
G
z mutant plasmid was transfected into HeLa cells, and after
20 hours the cells were processed for immunofluorescence
(Fig. 8). The triple
G
z mutant was mainly distributed in intracellular membranous
structures including those in the perinuclear region. Golgi protein membrin
was dispersed in 39% of the cells expressing the triple G
z
mutant, whereas it was not dispersed in wild-type
G
z-expressing cells. Taken together, the effects of
overexpression of RGS proteins and the dominant-negative G
z
mutant strongly suggest a critical role of active G
z in
maintenance of the Golgi structure.
|
Association of Gz with the Golgi apparatus
Previous studies showed that the expression of Gz is
limited primarily to platelets, neurons and chromaffin cells, suggesting
specific roles in these tissues (Matsuoka
et al., 1988
; Casey et al.,
1990
; Hinton et al.,
1990
). If G
z is involved in the organization of
the Golgi apparatus, it should be expressed ubiquitously. To demonstrate the
expression of G
z and to determine its localization in
non-neuronal cultured cells, we performed indirect immunofluorescence
microscopic analysis. Immunoreactivity to the anti-G
z
antibody was mainly observed in the perinuclear region, with some in the
plasma membranes of BHK and Clone9 cells
(Fig. 9a,c). The perinuclear
structure positive for the anti-G
z antibody most likely
corresponds to the Golgi apparatus, because it was also stained by medial
Golgi marker Man II (Fig.
9b,d). The staining with the anti-G
z antibody is
specific because it was totally abolished when the antibody was preincubated
with the peptide used for immunization
(Fig. 9e,g). Similar Golgi
labeling patterns for G
z were detected for other cells
including PC12, Chinese hamster ovary, and NRK cells (data not shown). To
confirm that the perinuclear G
z staining reflects the Golgi
structure, we treated cells with brefeldin A (BFA). BFA is known to cause the
redistribution of Golgi-resident proteins to the ER
(Klausner et al., 1992
). The
perinuclear G
z staining as well as the Golgi marker Man II
staining was dispersed upon the treatment of cells with BFA
(Fig. 9i-l), suggesting that
endogenous G
z is located in the Golgi apparatus.
|
Expression of Gz was also assessed by semi-quantitative
RT-PCR analysis (Fig. 10).
mRNA for G
z was expressed in non-neuronal Clone9 and NRK
cells, although the neuron-like PC12 cells showed much higher expression. The
control GAPDH mRNA level was approximately the same in these samples. The
ubiquitous expression of G
z is consistent with the recent
observation that G
z is detectable in various tissues of
mouse (Hendry et al.,
2000
).
|
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Discussion |
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When Gz-selective GAPs such as RGSZ1
(Glick et al., 1998
;
Wang et al., 1998
) and RGSZ2
were expressed, various Golgi-resident proteins including ß-COP and Man
II showed dispersed distribution patterns in several types of cells. In
contrast, the structures of the ER and microtubules were not markedly affected
by the expression of these proteins. No significant Golgi disassembly was
observed when RGS proteins other than RGSZ1 and RGSZ2 were expressed. These
results suggest that the inactivation of G
z but not other
G
proteins, such as G
i2, induces Golgi disassembly.
Chatterjee and Fisher demonstrated that RGSZ1, when expressed in COS-7 cells,
is localized in the Golgi apparatus
(Chatterjee and Fisher, 2000
).
In our experiments, expressed RGSZ1 was mainly distributed throughout the
cytoplasm and nucleus. This discrepancy can be partly explained by the use of
different cell lines. We also observed Golgi-like perinuclear localization of
exogenously expressed RGSZ1 in COS-7 cells. However, similar to in other cell
lines, a dispersed pattern of a Golgi marker ß-COP was observed in cells
expressing RGSZ1 at a high level (data not shown).
The idea that inactivation of Gz induces disassembly of
the Golgi apparatus was supported by another finding with the use of a
dominant-negative mutant, G
z(G204A/E246A/A327S). This mutant
was designed in analogy with a dominant-negative G
s mutant
in which three conserved residues are simultaneously mutated
(Iiri et al., 1999
). Since
this type of mutant remains in a guanine nucleotide-free form, it can inhibit
G
-mediated signaling, probably by occupying activated receptors. As
expected, overexpression of the triple G
z mutant induced
Golgi disassembly.
Although Gz is formally a member of the Gi
family, it possesses several unique biochemical properties distinct from those
of other G
i members
(Fields and Casey, 1997
). One
unique character of G
z is its insensitivity to pertussis
toxin-mediated ADP-ribosylation (Casey et
al., 1990
), a modification that inactivates other members of the
Gi family. In addition to general G-protein activators such as
GTP
S and AlF4-, mastparan, a peptide that selectively
activates the Gi family, also blocked the NDGA-induced Golgi
disassembly (data not shown). In fact, the overexpression of either
G
z or G
i2, both of which belong to the
Gi family, has a similar protective effect against NDGA-induced
Golgi disassembly (Yamaguchi et al.,
2000
). However, treatment of cells with pertussis toxin did not
affect the Golgi morphology (data not shown), suggesting the involvement of a
pertussis toxin-insensitive G
, i.e. G
z, in the
maintenance of the Golgi structure. The fact that RGS3 and RGS4, both of which
can attenuate G
i activity, lack Golgi disassembly activity
is consistent with this idea. Although overexpression of both
G
i2 and G
z can block NDGA-induced Golgi
disassembly, G
z may be involved in the maintenance of Golgi
structure under physiological conditions.
Based on the results reported here, we envisage a mechanism by which
Gz controls the structure of the Golgi apparatus. When
G
z binds GTP, it activates a signaling cascade that is
required for maintenance of the Golgi structure. When G
z is
inactivated upon the hydrolysis of bound GTP, the Golgi structure undergoes
disassembly as a consequence of loss of the signaling. Thus,
G
z functions as a molecular switch that organizes the Golgi
structure. This hypothesis predicts that G
z preferentially
binds GTP, thereby being constitutively active, in interphase cells. From this
point of view, G
z seems to be a favorable G protein because
it exhibits a very slow intrinsic rate of GTP hydrolysis. Its k cat
value is 200-fold lower than those of G
s and
G
i (Casey et al.,
1990
).
Investigation of the mechanism by which Gz maintains the
Golgi structure is a future challenge. The Golgi disassembly induced by
inactivation of G
z does not appear to involve fast release
of coat proteins (data not shown), as seen in cells treated with BFA. In
addition, expression of RGSZ1 does not affect microtubule organization.
G
z may act on Golgi stacking proteins or factors that can
link this organelle to the cytoskeletal elements. However, our results do not
exclude the possibility that G
z may control the export of
secretory and Golgi-resident proteins from the ER. The transport of VSVG-GFP
was partly inhibited in RGSZ1-expressing cells. Although our antibody failed
to detect it, a portion of G
z may exist in the ER.
Our present view that active Gz is required for
maintenance of the Golgi apparatus does not necessarily contradict the finding
by Malhotra and colleagues that the ß
dimer causes Golgi
disassembly (Jamora et al.,
1997
; Jamora et al.,
1999
). A similar situation was observed for trafficking from the
ER to the Golgi apparatus. The addition of ß
dimer, which leads to
inactivation of active G
by shifting the equilibrium toward the
formation of the trimeric complex, inhibits the formation of vesicles from the
ER in semi-intact cells. Mastparan, an activator for heterotrimeric G
proteins, also blocks vesicle formation
(Schwaninger et al., 1992
).
The presence of multispecies of G proteins in organelles may account for these
intricate and apparently inconsistent observations. It should be noted that
the expression of G
z in non-neuronal cells is not high. The
amount of the ß
dimer released as a consequence of the activation
of G
z must be very small, and therefore may not be enough to
cause Golgi disassembly. On the other hand, activation of major G proteins may
yield a large amount of the ß
dimer, which results in disassembly
of the Golgi apparatus. Gi family proteins may be more widely
involved in the regulation of organelle structures in early secretory
pathways. Wang et al. reported that treatment of rat hepatocytes with
pertussis toxin caused the redistribution and fragmentation of the ER
(Wang et al., 2000
). Thus,
different G proteins may be involved in more intricate cellular functions than
presently assumed.
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Acknowledgments |
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References |
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