GAIP, a Gai-3-binding protein, is associated with
Golgi-derived vesicles and protein trafficking
FIONA WYLIE, KIRSTEN HEIMANN, TAM LUAN LE, DARREN BROWN,
GLENN RABNOTT, AND JENNIFER L. STOW
Centre for Molecular and Cellular Biology, University of Queensland,
Brisbane, Queensland 4072, Australia
regulators of G protein signaling family of proteins; vesicle
trafficking; heterotrimeric G protein regulation
regulate a variety of signal
transduction pathways in eukaryotic cells, such as the
photoreception mechanism, hormone receptor signaling, ion channel regulation, and membrane trafficking
(4, 21, 48). In recent years, G protein subunits have
been localized on a variety of organelle membranes,
including the endoplasmic reticulum (1), the Golgi
complex (20), and endosomal structures (8). In addition, it has been demonstrated that vesicle trafficking
in the secretory pathway is regulated at multiple steps
by heterotrimeric G proteins (33, 42, 44, 49, 51). The
signal transduction pathways regulating vesicular
transport are not well defined. Effectors, receptors, and
other regulators of heterotrimeric G proteins in these
pathways remain to be characterized.
HETEROTRIMERIC G PROTEINS
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Ga-interacting protein (GAIP) is a member of the
recently described regulators of G protein signaling
(RGS) family (17, 30, 46). These proteins are able to
negatively regulate heterotrimeric G protein activity
through specific binding to Ga subunits. The RGS
functional prototype is Sst2 in yeast, a pheromonesensitive agent that negatively regulates the activity of
Gpa1 (6, 16, 18). There are at least 21 RGS proteins
already cloned and, with the exception of the cholerasensitive subunit Gsa, all other Ga subunits bind one or
more of the RGS proteins with varying affinities (38).
GAIP was identified in a yeast two-hybrid screen using
Gai-3 as the bait (14). In vitro, GAIP binds activated
forms of Gai-3 with high affinity and other members of
the Gai subfamily with varying but lower affinities.
GAIP and other RGS proteins have been shown to act
as GTPase-activating proteins (GAPs) for heterotrimeric G proteins; the kinetics of this activity differ
according to the binding affinity for the interacting Ga
subunit (3, 7, 52). RGS proteins are characterized by a
stretch of 125 amino acids (19, 46) (the RGS domain)
that interacts with the Ga subunit (14) and mediates
the GTPase activity of the RGS protein (43). In addition
to in vitro experiments, this GTPase activity has been
demonstrated in defined G protein pathways in intact
cells or microsomal fractions (19, 26, 39). Recently,
RGS9 was identified as a highly efficient GAP for Gat
(transducin) in the retina, where, in conjunction with
the cGMP phosphodiesterase effector, it regulates recovery of visual transduction (24).
GAIP thus has the potential to act as a powerful
regulator of Gai-3 signaling pathways. Due to the
localization of heterotrimeric G proteins on Golgi membranes and the demonstrated involvement of Gai-3 in
vesicular transport, the RGS protein GAIP was investigated as a possible player in the regulation of protein
trafficking. Data in two recent studies indicate that
GAIP is involved in vesicular trafficking. In colonic
epithelial cells, GAIP was demonstrated to regulate
trafficking in the autophagic sequestration pathway,
and the effects of overexpressing GAIP were consistent
with its function as a GAP for Gai-3 in this pathway (41).
Another recent report shows that, in pituitary and liver
cells, membrane-bound GAIP is localized on clathrincoated vesicles (13). The full inventory of trafficking
pathways involving GAIP now stands to be elucidated.
The locations and roles of GAIP may vary between cell
types, as do those of Gai-3. The specific membrane
locations of GAIP and other RGS proteins might therefore serve as a preliminary indication of their links with
specific Ga subunits and their potential roles in different steps in vesicle trafficking. In this paper, we have
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
C497
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Wylie, Fiona, Kirsten Heimann, Tam Luan Le, Darren
Brown, Glenn Rabnott, and Jennifer L. Stow. GAIP, a
Gai-3-binding protein, is associated with Golgi-derived vesicles
and protein trafficking. Am. J. Physiol. 276 (Cell Physiol. 45):
C497–C506, 1999.—Proteins of the regulators of G protein
signaling (RGS) family bind to Ga subunits to downregulate
their signaling in a variety of systems. Ga-interacting protein
(GAIP) is a mammalian RGS protein that shows high affinity
for the activated state of Gai-3, a protein known to regulate
post-Golgi trafficking of secreted proteins in kidney epithelial
cells. This study aimed to localize GAIP in epithelial cells and
to investigate its potential role in the regulation of membrane
trafficking. LLC-PK1 cells were stably transfected with a
c-myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP was found in the cytosol and on cell
membranes. Immunogold labeling showed that membranebound GAIP was localized on budding vesicles around Golgi
stacks. When an in vitro assay was used to generate vesicles
from isolated rat liver and Madin-Darby canine kidney cell
Golgi membranes, GAIP was found to be concentrated in
fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was
measured in cell lines overexpressing GAIP. We show evidence for GAIP regulation of secretory trafficking before the
level of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only
partially with those of Gai-3 and suggest multiple roles for
GAIP in epithelial cells.
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ROLE OF GAIP ON GOLGI VESICLES
investigated the distribution of GAIP on intracellular
membranes in kidney epithelial cells, its interactions
with membrane-bound Gai-3, and its involvement in
Gai-3-regulated trafficking in the secretory pathway.
MATERIALS AND METHODS
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Antibodies. Polyclonal antibodies were raised in both chickens and rabbits against human GAIP expressed as a glutathione S-transferase (GST) fusion protein. Animals were injected with glutathione-purified GST-GAIP protein. IgY
fractions were purified from chicken egg yolks (Eggstract,
Promega, Madison, WI), and rabbit sera were affinity purified
over cyanogen bromide-activated Sepharose conjugated to
the GST-GAIP fusion protein, and then antibodies were
additionally affinity purified on thrombin-cleaved GAIP peptide columns. Antisera were tested for reactivity and specificity by immunoblotting on GST-GAIP and on cell extracts (see
Fig. 1). An affinity-purified goat polyclonal antibody (C-20)
raised against a 20-residue GAIP peptide (197–216) was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
EC polyclonal antibody, raised against a COOH-terminal
peptide of the Gai-3 subunit, was purchased from DuPont
(Boston, MA). A monoclonal antibody specific for the c-myc
epitope tag was purchased from Promega. Indocarbocyanine
(Cy3)-conjugated rabbit- and mouse-specific antibodies were
supplied by Jackson Immunoresearch (West Grove, PA). AD7
monoclonal antibody, which recognizes p200/myosin II, was
raised and characterized as previously described (34). A
polyclonal antibody against the b-subunit of coatomer (bCOP) was purchased from Sigma-Aldrich.
Cell culture and transfection. LLC-PK1 cells (pig kidney
epithelial cells) were grown, passaged as confluent monolayers in DMEM containing 2% L-glutamine and 10% FCS, and
transfected as previously described (5, 49). Cells were transfected with cDNA encoding human GAIP, either ligated to a
c-myc expression tag (MEQKLISEEDLN) at the COOHterminal end and cloned into a pCB7 vector (Pharmacia
Biotechnology, Uppsala, Sweden) or cloned directly into a
pcDNA3.1 expression vector (Invitrogen, San Diego, CA).
Transfections were carried out using 10 µg cDNA and N-[1(2,3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposomal reagent (Boehringer Mannheim, Mannheim, Germany). LLC-PK1 cells transiently transfected with
c-myc-GAIP, untagged GAIP, or the pcDNA3.1 vector alone
were used for some experiments. Dishes of cells at 50%
confluence were transfected, and duplicate dishes were
screened by immunofluorescence to select dishes with 20%
transfection efficiency, which is routine for this cell line. Cells
were used at 24 h posttransfection for analyses. For stable
expression of GAIP, transfected cells were selected using
200–500 µg/ml hygromycin B (Boehringer Mannheim) and
cloned. Clones stably expressing c-myc-GAIP at levels of 2- to
20-fold were screened and selected; these were denoted
GMLLC-PK1 cells. GMLLC-PK1 clones 7 and 1 were used for
all experiments reported here; these clones were determined
by immunoblotting with titrated antibodies and by immunofluorescence to be expressing GAIP at 5- and 20-fold over
endogenous levels of GAIP, respectively.
SDS-PAGE and immunoblotting. Protein mixtures to be
analyzed were separated by SDS-PAGE on 12% Laemmli
denaturing gels under reducing conditions. For immunodetection, the proteins were electrotransferred onto polyvinylidene
difluoride Immobilon P membrane (Millipore, Bedford, MA)
and stained in 0.1% Coomassie blue (in 50% methanol and
10% acetic acid) to check protein loading. Membranes were
blocked and washed in Blotto solution (20 mM Tris · HCl
buffer, pH 7.5, containing 0.15 M NaCl, 0.1% Triton X-100,
and 5% nonfat milk ) before an overnight incubation at 4°C in
primary antibody. Bound specific antibody was detected by
incubation using an alkaline phosphatase-conjugated secondary antibody (1:1,000) and the substrate 5-bromo-4-chloro-3indolylphosphate (Sigma-Aldrich).
Immunofluorescence. Cells grown as preconfluent monolayers on glass coverslips were washed once in PBS (pH 7.4)
before fixing in either 4% paraformaldehyde for 1 h at room
temperature or in cold methanol-acetic acid (3:1) for 10 min at
220°C. The cells were permeabilized in 0.1% Triton X-100 in
PBS and then blocked in 0.5% BSA diluted in PBS. Primary
antibody incubations were carried out for 1–2 h at room
temperature and detected using a Cy3-conjugated secondary
antibody. The coverslips were mounted in 1% N-propyl gallate
in 50% glycerol and viewed on an Olympus Provis AX70
microscope.
Cell fractionation. Confluent cell monolayers were scraped
into homogenization buffer (10 mM Tris · HCl, pH 7.2, containing 1 mM EDTA) and left for 5 min at room temperature. The
cells were homogenized in 0.5-ml lots by 20 passages through
a 27.5-gauge needle and then centrifuged at 1,000 g for 10
min at 4°C. The postnuclear supernatant was centrifuged at
100,000 g for 90 min. The pellet was resuspended in homogenization buffer; this constituted the total microsomal membrane fraction. The supernatant was recentrifuged at 100,000
g for 45 min to remove membranes, and this supernatant was
collected as the cytosol fraction. Protein estimations (Pierce,
Rockford, IL) and SDS-PAGE analysis were carried out on all
fractions.
Golgi vesicle budding assay. A Golgi membrane fraction
was prepared from homogenates of rat liver or Madin-Darby
canine kidney (MDCK) cells by density gradient centrifugation, based on the method of Leelavathi et al. (31). Golgi
membranes were incubated in vitro with cytosol (1 mg/ml
protein), 100 µM guanosine 58-O-(3-thiotriphosphate)
(GTPgS), and a modified HKM buffer [in mM: 25 HEPES (pH
7.2), 20 KCl, and 2.5 Mg(CH3COO)2 (45)] containing 0.5 mM
dithiothreitol and an ATP-regenerating system (4.6 IU/ml
creatine phosphokinase, 81 mM creatine phosphate, and 28.6
mM ATP) to generate budded vesicles (10). Budded vesicles
and remaining Golgi cisternae were separated by centrifugation at 17,500 g for 10 min. The vesicles in the supernatant
were then separated from the remaining cytosol by ultracentrifugation at 100,000 g for 90 min. Fractions were analyzed
by SDS-PAGE and immunoblotting using antibodies to vesicleassociated proteins (10) and to GAIP.
Immunogold labeling. Cells for cryosectioning were fixed in
4% paraformaldehyde-0.1% glutaraldehyde (pH 7.4) for 1 h,
scraped off the culture dish, and pelleted in 2% gelatin.
Sections (80 µm) collected on Formvar/carbon-coated copper
grids were blocked in 1% BSA in PBS and then incubated in
primary antibody for 1 h. Bound antibody was visualized
using protein A conjugated to 10-nm colloidal gold particles
(Dr. J. W. Slot, Dept. of Cell Biology, Utrecht, The Netherlands). Sections were contrasted and embedded in 1% uranyl
acetate-2% methylcellulose on ice for 10 min and viewed in a
JEOL 1010 microscope at 80 kV. Perforated MDCK cells were
prepared from monolayers plated at confluence on 24-mm
Transwell filters (Corning Costar, Cambridge, MA) (28).
Briefly, the filters were incubated at 20°C for 20 min, washed,
and partially dried, and then the apical membranes were
perforated by application and removal of a nitrocellulose
filter. The filters were incubated at 37°C for 15 min in wash
buffer [in mM: 25 HEPES-KOH (pH 7.2), 2.5 Mg(CH3COO)2,
50 KCH3COO, 5 EGTA, and 1.8 CaCl2] containing cytosol to 1
mg/ml, aluminum fluoride (AlF2
4 : 50 µM AlCl3, 30 mM NaF),
ROLE OF GAIP ON GOLGI VESICLES
RESULTS
GAIP is found in membrane and cytosol cell fractions.
Affinity-purified chicken and rabbit antibodies raised
against a GST-GAIP fusion protein were shown to
recognize the fusion protein and a single, 29-kDa band,
corresponding to GAIP, in cell extracts (Fig. 1); this
same band was also recognized by an antibody (C-20)
raised against a COOH-terminal peptide of GAIP (Fig.
1A). The chicken and rabbit antibodies did not recognize bands corresponding to other proteins or other
known RGS proteins in these extracts and were determined to be specific for GAIP. The antibodies were used
to probe for endogenous GAIP in a number of cell lines.
Cell extracts were fractionated into total microsomal
membranes and cytosol and immunoblotted for GAIP
using the chicken antibody; four of these are shown in
Fig. 1B. Endogenous GAIP was expressed at roughly
equivalent, relatively low levels in all cells tested. In
cultured epithelial cell lines, GAIP was detected pre-
Fig. 1. Immunoblotting of endogenous and recombinant Ga-interacting protein (GAIP). A: preparations of glutathione S-transferase
(GST)-GAIP fusion protein (6 µg/lane) and GMLLC-PK1 (clone 1)
cytosol (12 µg total protein/lane) were blotted with a commercial
antibody (C-20) or with chicken or rabbit antibodies raised against a
GST-GAIP fusion protein (all used at 1:1,000 dilution). All antibodies
recognized the same 56-kDa fusion protein band. A single band
corresponding to recombinant GAIP (,29 kDa) in cytosol preparations was recognized by C-20 and rabbit GAIP antibodies. B: membrane (M) and cytosol (C) fractions prepared from LLC-PK1, Chinese
hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and
macrophage (RAW 264) cell lines were immunoblotted with chicken
GAIP antibody. Endogenous GAIP was detected as a 29-kDa band in
these fractions, although GAIP in RAW membranes migrated slightly
faster. The antibody also reacted more weakly with an additional,
unidentified slower migrating band (*) in LLC-PK1 cells. C: GMLLCPK1 (clone 1) cells, stably transfected with c-myc-tagged GAIP (i) or
LLC-PK1 transiently transfected with untagged GAIP (ii), were
fractionated into membranes (M) and cytosol (C) and immunoblotted
using chicken antibody or a monoclonal antibody against c-myc
epitope tag. In GMLLC-PK1 cells, c-myc-tagged GAIP is detected in
both membrane and cytosol fractions as a band migrating slightly
slower than 29 kDa due to presence of epitope tag. Some endogenous
GAIP, migrating as faster bands, is also detected in cytosol by GAIP
antibody. Untagged GAIP is also expressed, as a 29-kDa band, in
membranes and cytosol of transiently transfected LLC-PK1 cells.
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and an ATP-regenerating system (1 mM creatine phosphate,
8 U/ml creatine phosphokinase, and 50 µM ATP-Na). The
filters were then fixed in 0.1% glutaraldehyde-4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at room
temperature; strips were excised from the filter and immunogold labeled as described above. The filter strips were then
postfixed for 1 h in 2.5% glutaraldehyde in 0.1 M sodium
cacodylate containing 0.1 g/ml sucrose, 12.5 mM CaCl2, 70
mM KCl, and 12.5 mM MgCl2, followed by 1% osmium
tetroxide in potassium ferrocyanide for 30 min. The samples
were then stained en bloc in 0.5% uranyl acetate in 50%
ethanol, dehydrated through a graded series of ethanols, and
embedded in Epon 812 resin. Ultrathin sections were cut
(Reichardt Ultracut S), contrasted in lead acetate and uranyl
acetate, and viewed as described above.
Secretion of sulfated proteoglycan. GMLLC-PK1 (clones 1
and 7), untransfected LLC-PK1 cells, and transiently transfected LLC-PK1 cells were plated at confluence on 24-mm
Transwell filters (Corning Costar) and used to measure the
sulfation and secretion of [35S]sulfate-labeled basement membrane heparan sulfate proteoglycan (bmHSPG), as described
previously (11, 49). Transiently transfected cells with equivalent levels of transfection were plated in triplicate wells for
each experiment. Cells were washed and preincubated in
modified sulfate-free Fischer’s medium (GIBCO BRL, Grand
Island, NY) and then metabolically labeled for 3 h at 37°C
using 500 µCi/ml [35S]sulfate (carrier free; ICN Biomedical)
added to the basal medium. Duplicate filters were also
labeled with [35S]cysteine (500 µCi/ml; to measure total
bmHSPG synthesis and secretion) or with [35S]sulfate in the
presence of 1 mM b-D-xyloside to measure total 35S uptake
into glycosaminoglycans. After the labeling, the apical and
basal media were collected and the cells were scraped off the
filters into RIPA buffer (25 mM Tris · HCl, pH 7.4, containing
1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 0.15 M
NaCl). The bmHSPG was immunoprecipitated from each of
the fractions, immunoprecipitates were run on 5–15% gradient SDS-PAGE gels, and [35S]bmHSPG in intracellular (cell
layer) and secreted pools (apical or basal media) were compared by fluorography and densitometry (11).
Immunoprecipitation of GAIP and Gai-3. Whole cell homogenates or total membrane fractions from GMLLC-PK1 cells
were extracted by incubation for 10 min at room temperature
in HKM buffer (30 mM HEPES, pH 7.4, with 20 mM KCl and
5 mM magnesium acetate) containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Extracts were then centrifuged at 14,000 g for 10 min at 4°C to
remove unextracted material. Incubations and washes were
carried out in HKM buffer with 1% CHAPS. The supernatant
was incubated overnight at 4°C with EC antibody, raised
against Gai-3. Protein A-Trysacryl beads (Pierce) resuspended
in 10 mM Tris · HCl (pH 7.4) were added to the supernatant
and incubated for 1 h at room temperature with constant
mixing. After brief centrifugation, the supernatant was collected as the depleted starting material and the beads were
collected as the immunoprecipitate. The beads were washed
several times in excess 10 mM Tris · HCl (pH 7.4), and finally
immunoprecipitated proteins were solubilized in SDS-PAGE
sample buffer. Samples were analyzed by SDS-PAGE and
immunoblotted using both EC and GAIP-specific antibodies.
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ROLE OF GAIP ON GOLGI VESICLES
Fig. 2. Localization of GAIP by immunofluorescence. Immunostaining of overexpressed GAIP revealed a diffuse cytosolic distribution
with a perinuclear concentration of GAIP staining in some cells.
GMLLC-PK1 (clone 1) cells were labeled with anti-c-myc (A; 1:100)
and chicken anti-GAIP (B; 1:500).
In double-labeling experiments, GAIP-labeled vesicles
were not colabeled for p200/myosin II, which is found
on a population of trans-Golgi network (TGN)-derived
vesicles (27) (data not shown). The low level of GAIP
labeling in these sections was consistent with the
relatively small amount of membrane-bound endogenous GAIP detected by immunoblotting in MDCK cell
fractions. In all, immunostaining showed a diffuse
distribution of GAIP throughout the cytoplasm and
specific labeling of membrane-bound GAIP on a population of Golgi-derived vesicles.
Fractionation of Golgi vesicles from rat liver and
MDCK cells. Density gradient centrifugation was used
to further study the association of GAIP with Golgiderived vesicles. Rat liver Golgi membranes and cytosol
from rat liver homogenates and MDCK cells were used
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dominantly (.90%) in the cytosol fractions with much
smaller amounts in the membrane fractions. In macrophages (RAW 264.7), a band of slightly faster mobility
was recognized by the GAIP antibody in membrane
fractions. This shift in electrophoretic mobility could
reflect conformational differences between cytosolic
and membrane-bound forms of GAIP due to posttranslational modifications such as palmitoylation (12).
LLC-PK1 cells were stably transfected with a c-myctagged GAIP cDNA (Fig. 1C) or transiently transfected
with an untagged GAIP construct (Fig. 1C). Stably
transfected clonal cell lines expressing different levels
of c-myc-GAIP were used in subsequent experiments
and are referred to as GMLLC-PK1 cells. Immunoblots
of cell extracts labeled with antibodies against either
GAIP or the c-myc tag showed the presence of recombinant, c-myc-tagged GAIP in both the cytosol and membrane fractions of stably transfected GMLLC-PK1 cells.
Transient transfection also resulted in expression of
recombinant GAIP in both cytosol and membrane fractions. As in untransfected cells, most of the overexpressed GAIP was found in the cytosol (60–70%),
although a proportion (30–40%) of recombinant GAIP
was found also in membrane fractions. Both untagged
GAIP (Fig. 1Cii) and c-myc-GAIP (Fig. 1Ci) showed the
same distribution in cytosol and membrane fractions.
Localization of GAIP in cells. Immunofluorescence
staining, carried out on a variety of cell lines and
transfected cells, showed distributions of GAIP similar
to that found by immunoblotting. GMLLC-PK1 cells
overexpressing c-myc-tagged GAIP showed predominantly cytoplasmic staining (Fig. 2). Although a heavier
concentration of GAIP staining was often observed in
the perinuclear region (Fig. 2A), no specific staining of
any intracellular membranes or organelles, or any
staining of the cell surface, was detected at this level.
The more sensitive method of immunogold labeling at
the electron microscopy level also was used to localize
GAIP. Ultrathin cryosections of GMLLC-PK1 cells
showed c-myc-GAIP labeling throughout the cytoplasm
(Fig. 3A). Again, no concentrated immunogold labeling
of plasma membrane or of other organelles was obvious
in the cryosectioned cells, perhaps due to the abundant
cytoplasmic staining. Immunogold labeling was therefore also carried out on perforated MDCK cells from
which the bulk of the cytosolic proteins had been
removed, leaving only intracellular organelles and the
proteins associated specifically with them (Fig. 3, B–D).
Specific gold labeling of endogenous GAIP was found
predominantly adjacent to membranes, showing the
effective removal of cytosolic GAIP. Membrane labeling
of GAIP was seen on a proportion of vesicles in the
vicinity of Golgi stacks and not on any other membranes or organelles in MDCK cells. GAIP labeling was
commonly on newly budded vesicles and on the budding
ends of Golgi cisternae, with only occasional labeling on
Golgi cisternal membranes. The specific population of
GAIP-labeled vesicles could not be identified in these
samples. Some, but not all, of the vesicles had electrondense coats, and yet other vesicles, with identifiable
electron-dense, clathrin coats, were not labeled (Fig. 3).
ROLE OF GAIP ON GOLGI VESICLES
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to reconstitute in vitro vesicle budding in the presence
of GTPgS (10). The distribution of GAIP was compared
with other peripheral, vesicle-associated proteins, p200/
myosin II and b-COP, which are known to bind to
distinct populations of Golgi-derived vesicles under
these conditions (10, 35). In this assay, b-COP and
p200/myosin II derived from the cytosol bind to vesicles
in a GTP-dependent fashion (Fig. 4, lane 3). Most of the
GAIP was also initially found in the cytosol; it was not
detected in the freshly isolated stacked-Golgi membrane fraction (Fig. 4, lane 2). After incubation, GAIP
was enriched in budded vesicle fractions (lanes 3 and 6)
but was not present in significant amounts on remnant
Golgi cisternae (lane 4). This pattern suggests that
GAIP binds to vesicles during budding and is not
retained on cisternal membranes. These results are
consistent with the immunogold labeling in showing
that GAIP is found primarily on Golgi-derived vesicles
and that little, if any, GAIP appears to reside on Golgi
cisternae. These data also highlight the temporal segre-
gation of GAIP and Gai-3. As can be seen in Fig. 4, all of
the Gai-3 is membrane bound, and it resides on the
Golgi cisternal membranes (lanes 2 and 4). After budding, some of the Gai-3 fractionates with the vesicles
(lane 6), and this is the only site where GAIP and Gai-3
may be colocated.
Membrane-bound GAIP associates with activated
Gai-3. The partial overlap of Gai-3 (on Golgi membranes
and vesicles) and GAIP (in the cytosol and on vesicles)
raises the question of whether or not these two proteins
do interact in cells. In assays using purified components, RGS proteins have been shown to bind specifically to Ga subunits and with highest affinity to a
transitionally activated state of Ga, induced by the
21 (2).
addition of AlF2
4 and GDP in the presence of Mg
We tested the ability of c-myc-GAIP expressed in
GMLLC-PK1 cells to bind to Gai-3, which is found on the
Golgi membranes in these cells (49). Gai-3 was immunoprecipitated from both whole cell extracts and mem-
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Fig. 3. Ultrastructural localization of
GAIP. A: cryosections of GMLLC-PK1
(clone 1) cells were labeled with anti-cmyc (1:10) and 10-nm protein A-gold;
labeling was distributed throughout cytoplasm, with some gold labeling in
areas of tubular vesicular membranes,
although there was no specific gold
labeling on any organelles. B–D: ultrathin resin sections of perforated MDCK
cells, labeled before embedding with
rabbit anti-GAIP (1:10) and protein Agold. Although cytosol has been removed from these cells, individual organelles are still intact. These fields
depict perinuclear areas, including
Golgi stacks. Gold labeling (arrowheads) was seen on vesicles and some
tubular structures in the vicinity of
Golgi stacks (G) and sometimes on budding ends of cisternae (arrow, micrograph C). Scale bars, 0.2 µm.
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ROLE OF GAIP ON GOLGI VESICLES
Fig. 4. Fractionation of rat liver Golgi membranes. Isolated Golgi
membranes from rat liver or MDCK cells were incubated with cytosol
fractions in a vesicle budding assay. Top: samples corresponding to
original total Golgi membranes (lane 1; 20 µg protein) and cytosol
fractions (lane 2; 100 µg protein), budded vesicles (lanes 3 and 6; 10%
of supernatant and 20 µg protein, respectively), remnant Golgi
cisternae (lane 4; 20 µg protein), and final supernatant of remaining
cytosol (lane 5; 10% of supernatant) were analyzed by SDS-PAGE and
immunoblotted with chicken anti-GAIP (1:100). Rat liver samples
were also analyzed for other vesicle-associated proteins using antibodies to p200/myosin (1:100), b-COP (1:100), and Gai-3 (1:1,000). For
both rat liver and MDCK cells, GAIP was detected in original cytosol
fractions but not in total Golgi membranes. After vesicle budding,
GAIP was found predominantly on budded vesicles in lanes 3 and 6,
with a very small amount on remnant Golgi cisternae (lane 4). A
significant amount of GAIP was now detected in budded vesicles (lane
6). Bottom: relative amounts of GAIP recovered in each lane (total
GAIP added to assay 5 100%). These data are representative of 6
separate fractionations and budding assays for both rat liver and
MDCK cells.
brane fractions of GMLLC-PK1 cells in buffers containing combinations of Mg21, AlF2
4 , and GDP (Fig. 5). The
41-kDa Gai-3 was immunoprecipitated under all conditions from these samples. Immunoprecipitates were
analyzed by immunoblotting to detect coprecipitated
GAIP. Only small amounts of c-myc-GAIP were coprecipitated with Gai-3 from untreated cell extracts or in
the presence of added Mg21 and AlF2
4 alone. The
Fig. 5. Coprecipitation of GAIP and Gai-3. A: GMLLC-PK1 (clone 1)
whole cell extracts (200 µg total protein) were incubated with
combinations of Mg21, AlF2
4 , and GDP, and then immunoprecipitated
with Gai-3 antibody. Precipitates (IP; 100% of sample) and supernatants (S; 5% of sample) were analyzed by SDS-PAGE and immunoblotted for GAIP (chicken antibody; 1:1,000) and Gai-3 (1:1,000).
Association of GAIP and Gai-3, seen as presence of a large amount of
GAIP in Gai-3 precipitate, was strongest in presence of AlF2
4 and GDP.
B: coprecipitation of GAIP and Gai-3 with Gai-3 antibody was compared in 200 µg samples of GMLLC-PK1 (clone 1) whole cell extract
and GMLLC-PK1 (clone 1) membranes, in the presence of AlF2
4 and
GDP. Gel fragments shown represent total precipitated protein.
GAIP from both fractions was coprecipitated with Gai-3 but relatively
more membrane-bound GAIP was associated with Gai-3.
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coprecipitation of GAIP with Gai-3 was substantially
enhanced in the presence of both AlF2
4 and GDP,
suggesting a specific and conformation-dependent interaction between these two proteins (Fig. 5A). To decipher whether this bound GAIP was derived primarily
from the cytosol or from the smaller membraneassociated GAIP pool, Gai-3 was immunoprecipitated
from separate extracts of membranes or whole cell
lysates (Fig. 5B). Quantitation of coprecipitated proteins revealed that Gai-3 bound a higher proportion of
GAIP from its membrane-bound pool (,30%) than of
GAIP from the whole cell extract (,5%), which is
mostly cytosolic. These results suggest that, in cells,
membrane-bound GAIP can bind to the transitional
activation state of Gai-3 and that GAIP and Gai-3 are
unlikely to sustain this interaction throughout the
whole GTP-GDP cycle.
ROLE OF GAIP ON GOLGI VESICLES
Fig. 6. Effect of GAIP overexpression on secretion of basement
membrane heparan sulfate proteoglycan (bmHSPG). Trafficking of
constitutively secreted proteoglycan, bmHSPG, was studied in two
GMLLC-PK1 cell clonal lines, clone 1 (20-fold overexpression) and
clone 7 (5-fold overexpression). LLC-PK1 cells transiently transfected
(,20% cells transfected) and expressing equivalent levels of GAIP or
vector alone (pcDNA3.1) were also used in triplicate wells. Intracellular and secreted bmHSPG were labeled with [35S]sulfate and recovered and analyzed by immunoprecipitation, SDS-PAGE, and fluorography of cell extracts and basal medium samples. Relative amounts
of [35S]sulfate-labeled bmHSPG in cell extracts are represented as %
of control levels in untransfected LLC-PK1 cells. Decreased levels of
[35S]sulfate-labeled bmHSPG were found in clone 7 (5-fold overexpression: ,50% sulfation) and clone 1 (20-fold overexpression; ,12%
sulfation) GMLLC-PK1 cells, in proportion to their levels of GAIP
overexpression. Transient transfection of vector alone had no effect
on levels of sulfation or secretion. Transient transfection of cells with
GAIP resulted in reduced levels (80%) of sulfated bmHSPG, consistent with levels of GAIP transfection and expression. Relative
amounts of secreted (solid portions of bars) and intracellular (hatched
portions of bars) sulfated bmHSPG are shown. Despite altered
amounts of total [35S]bmHSPG in each sample, the proportion
secreted by each cell line remained constant, suggesting that postsulfation trafficking was not affected. These data are representative of 4
separate labeling experiments.
fate bmHSPG was present in transfected cells than in
the controls. Because [35S]sulfate is added to the
bmHSPG at the level of the TGN, these results imply
that overexpression of GAIP significantly retards delivery of bmHSPG precursors to the TGN, i.e., that
trafficking in earlier steps of the secretory pathway is
retarded. Sulfation function in GMLLC-PK1 cells was
tested in the presence of b-D-xyloside acceptor and was
found not to be compromised by GAIP overexpression
(data not shown). GAIP had little or no detectable effect
on post-TGN secretion of [35S]sulfate-labeled bmHSPG
into the basal medium of GMLLC-PK1 cells. The proportion of [35S]bmHSPG in cell layers and in medium was
the same in GMLLC-PK1 cells and control cells. Also,
the polarity of bmHSPG secretion (90% basal; 10%
apical) was unchanged in GMLLC-PK1 cells. Together
these data suggest that overexpression of GAIP does
not affect Gai-3-regulated post-TGN constitutive secretion of bmHSPG. However, the data are consistent with
overexpressed GAIP playing a significant role in Golgi
trafficking before the level of the TGN.
DISCUSSION
The intracellular distribution of GAIP and its potential participation in protein trafficking were investigated in kidney epithelial cells. Our current studies on
endogenous GAIP in a variety of cells and on recombinant GAIP in transfected LLC-PK1 cells indicate that
GAIP exists in both membrane-bound and cytosolic
pools. The majority of endogenous GAIP was found as a
soluble pool in the cytoplasm, and relatively small
amounts were found on membranes. In our hands,
transfected cells expressed proportionately more (10–
30% more) membrane-bound GAIP than untransfected
cells, suggesting that membrane binding sites for GAIP
are not saturated at steady state. This may lead to, and
be reflected in, differences in the relative levels of
membrane-bound and cytosolic GAIP measured at different times. De Vries et al. (12) showed variable
amounts of membrane-bound GAIP in different cell
lines; only 30% of overexpressed GAIP was in membrane fractions in transfected COS cells, whereas .80%
of GAIP in AtT20 cells was in the membrane fraction,
which coincided with heavy and predominant labeling
of GAIP on membranes in pituitary cells (13). The
differences observed, between this and previous studies
(12, 13), in the relative distribution of GAIP between
membrane and cytosol might reflect differences in the
cell types used or be due to cycling of GAIP between the
two pools. The relationship between pools of membranebound and cytosolic GAIP remains unclear at this
stage. GAIP has been shown to be modified for membrane attachment through palmitoylation at a number
of cysteine residues near the NH2 terminus (12), and
palmitoylation in other proteins, such as Gsa subunits,
provides a basis for their transient and regulated
attachment to membranes (32). GAIP may move dynamically between the two sites, as is suggested by the
GTP-dependent attachment of GAIP to Golgi membranes in the vesicle budding assay.
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Overexpression of GAIP retards constitutive secretory
trafficking. We and others have previously measured
the constitutive trafficking and secretion of [35S]sulfatelabeled bmHSPG to assay regulatory effects of Ga
subunits on vesicular trafficking (5, 33, 49). Proteoglycans, such as bmHSPG, are heavily sulfated at the level
of the TGN. In LLC-PK1 cells, we have shown that the
intracellular trafficking of bmHSPG can be retarded at
pre- and post-TGN steps by overexpression of Gai-3
(5, 49). A similar assay was used in this study to
investigate a functional role for GAIP by measuring the
effects of GAIP overexpression on constitutive trafficking in GMLLC-PK1 cells. Untransfected LLC-PK1 cells,
GMLLC-PK1 cells, and LLC-PK1 cells transiently transfected with vector only were labeled with [35S]sulfate;
radiolabeled bmHSPG precursors in cell extracts and
secreted bmHSPG, collected from the apical and basal
media, were analyzed by SDS-PAGE, fluorography, and
densitometry (Fig. 6). Compared with control LLC-PK1
cells and those transfected with vector alone
(pcDNA3.1), all GMLLC-PK1 cells produced significantly lower amounts of sulfated bmHSPG (Fig. 6),
although equivalent levels of early bmHSPG precursors were produced in all cell lines, as determined
by [35S]cysteine labeling of bmHSPG (data not
shown). Overexpression of GAIP in stably transfected
GMLLC-PK1 cells reduced [35S]sulfate bmHSPG in a
dose-dependent manner; two- to nine-fold less [35S]sul-
C503
C504
ROLE OF GAIP ON GOLGI VESICLES
apparently depend on Gai-3 interaction, since vesicles
are produced in the presence of GTPgS and GAIP does
not bind with high affinity to Gai-3-GTPgS (Fig. 5) (2).
Any interaction of Gai-3 on Golgi membranes with GAIP
on vesicle membranes would be most likely to occur
during the process of vesicle budding.
Studies using fusion proteins (3, 14, 52) have previously demonstrated high-affinity binding of GAIP to
the Gai-3 subunit, and one very recent study has now
confirmed that recombinant GAIP binds to Gai-3 in
intestinal cells (41). We also showed that c-myc-GAIP
in extracts of overexpressing GMLLC-PK1 cells is coprecipitated with Gai-3. Interaction occurred with highest
affinity in the presence of AlF2
4 and GDP, which emulates the transitional state on the Ga subunit. The GAP
function of RGS proteins has been attributed to the
ability of these proteins to interact with, and stabilize,
this transitional form (2). Our experiments additionally show that the membrane-bound form of GAIP, and
not that in the cytosol, accounts for the majority of
binding to Gai-3. Soluble forms of GAIP and Gai-3 must
be able to interact, since their binding in solution has
been demonstrated and GAIP was originally identified
as a soluble binding partner for Gai-3 in the yeast
two-hybrid system (14). It is likely that in intact cells
GAIP-Gai-3 interactions do occur at the membrane,
since Gai-3 in cells is always tightly bound to membranes (5). In the context of intact cells, it is also
possible that intervening protein interactions might
either enhance or prevent Gai-3 interactions with membrane-bound or cytosolic pools of GAIP.
Gai-3 has been demonstrated to regulate protein
trafficking in a variety of pathways (5, 33, 40, 41, 49,
53). We have previously shown that in LLC-PK1 cells
overexpression of Gai-3 subunits downregulates polarized (basolateral) constitutive secretion (49). The assay
used for these studies measures the trafficking of
precursors and the secretion of terminally sulfated
bmHSPG from polarized LLC-PK1 cells. This same
assay was applied here to cells overexpressing GAIP.
GAIP overexpression reduced the amount of [35S]sulfatelabeled bmHSPG in cells, although levels of [35S]cysteine-labeled bmHSPG precursors were unchanged,
suggesting that trafficking of bmHSPG before the level
of the TGN, where sulfation occurs, was retarded. GAIP
overexpression, even at high levels, did not alter the
kinetics of post-TGN secretion of bmHSPG. GAIP is
thus implicated in regulating constitutive trafficking
early in the secretory pathway. This correlates with one
of the stages of trafficking affected by overexpression of
Gai-3, which retards trafficking of bmHSPG precursors
in the pre-TGN Golgi (by ,3-fold). Overexpression of
Gai-3 also significantly reduces post-TGN secretion of
[35S]sulfate-labeled bmHSPG (5, 49), a step that is not
affected by GAIP. Thus GAIP appears to function in
only one of the steps of constitutive trafficking similarly
regulated by Gai-3. The known G protein-coupled functions for GAIP, to date, are as a GAP and as a potential
effector antagonist (25, 54). In the current assay, GAIP
did not downregulate the Gai-3 response, as would be
expected for GAP activity. The retardation of secretory
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In kidney epithelial cell lines, immunolocalization
and cell fractionation studies revealed that GAIP is
attached to vesicles associated with the Golgi complex.
Labeling of the relatively small pool of membranebound GAIP cannot be distinguished at the immunofluorescence level or by immunogold labeling on transfected cells, where staining of the abundant cytosolic
GAIP dominated. Membrane-bound GAIP was localized by immunogold labeling in perforated MDCK cells.
The use of perforated MDCK cells has proven useful,
and sometimes uniquely successful, for immunogold
labeling of peripheral proteins bound to the cytoplasmic face of Golgi membranes or vesicle membranes (22,
27, 28). Labeling of membrane-bound GAIP was restricted to a subpopulation of vesicles or budding
membrane structures around the Golgi stack. This
distribution indicates that GAIP associates selectively
with only some of the budding vesicles; similar distributions (on selected subpopulations of vesicles) are seen
for other vesicle-associated proteins, such as p200/
myosin II, b-COP, and g-adaptin, in these preparations.
Further characterization of these vesicle populations is
needed to identify the particular vesicles binding GAIP
in MDCK cells. De Vries et al. (13) have shown localization of GAIP on clathrin-coated vesicles, both those
budding from the Golgi and from the plasma membrane, in pituitary cells and liver cells. The vesicles
labeled here in MDCK cells could include Golgi-derived
clathrin-coated vesicles, although not all labeled vesicles
had recognizable coats and no plasma membranederived vesicles were labeled in perforated cells. The
paucity of labeling on Golgi cisternae, together with the
very small amount of GAIP detected in rat liver Golgi
cisternae fractions (Fig. 4), suggests that GAIP associates with the vesicle membranes only during or after
budding and that it does not reside on Golgi membranes.
Just how the localization of GAIP on these Golgi
vesicles relates to its interaction with Gai-3 is not clear.
The presence of both GAIP and Gai-3 on Golgiassociated membranes hints at a Golgi-specific functional link between the two proteins. Gai-3 is found on
the Golgi membranes of many cell types by immunofluorescence labeling (reviewed in Ref. 50); in LLC-PK1
cells, Gai-3 is exclusively located on Golgi membranes
(20, 49). At an ultrastructural level, Gai-3 appears
across the Golgi stack in LLC-PK1 cells (49), and in
exocrine pancreatic cells Gai-3 is also found from cis- to
trans-Golgi and on vesicles at both sides of the Golgi
stack by immunogold labeling (15). Studies in several
cell types show that Gai-3 regulates multiple pre- and
post-Golgi trafficking steps, suggesting that it may
function at multiple and variable sites across the Golgi
stacks of different cells (5, 33, 49, 53). Current and
previous data (13) showing that membrane-bound GAIP
is restricted to budded vesicles seemingly limit the
overlapping locations of Gai-3 and GAIP, and their
potential to interact, within the Golgi milieu. Coimmunoprecipitation of GAIP with Gai-3 confirmed that indeed the two proteins do, at some time, interact. The
binding of GAIP to Golgi vesicle membranes does not
ROLE OF GAIP ON GOLGI VESICLES
We thank Brandon Sullivan and Dennis Ausiello (Massachusetts
General Hospital, Harvard Medical School) for providing the GAIP
cDNA constructs and for helpful discussions and comments on the
manuscript.
This work was supported by a grant from the National Health and
Medical Research Council of Australia (to J. L. Stow).
J. L. Stow is a Wellcome Trust Senior Research Fellow.
Address for reprint requests: J. L. Stow, Centre for Molecular and
Cellular Biology, University of Queensland, Brisbane, QLD 4072,
Australia.
Received 16 July 1998; accepted in final form 2 November 1998.
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