Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
The assembly in living cells of heterotrimeric
guanine nucleotide binding proteins from their constituent ,
, and
subunits is a complex process, compounded by the multiplicity of the genes that encode
them, and the diversity of receptors and effectors with
which they interact. Monoclonal anti-
antibodies
(ARC5 and ARC9), raised against immunoaffinity purified
complexes, recognize
subunits when not associated with
and can thus be used to monitor assembly of
complexes. Complex formation starts
immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol,
and association of
complexes with the plasma membrane fraction starts between 15-30 min of chase. Three
pools of
subunits can be distinguished based on their
association with
subunits, their localization, and their detergent solubility. Association of
and
subunits
with detergent-insoluble domains occurs within 1 min
of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase.
Brefeldin A treatment does not interfere with delivery of
subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of
intracellular membrane trafficking and may occur directly at the plasma membrane.
Heterotrimeric guanine nucleotide binding proteins (G proteins)1 serve two major functions in
eukaryotic cells. Foremost is their role in signal
transduction, which comprises the interaction of a ligandbound heptahelical receptor with the intracellular machinery, such that signals delivered extracellularly result in the
appropriate cellular response, which is dependent on the
downstream effector system employed (19, 56). Heterotrimeric G proteins may also function as regulatory elements in membrane traffic (5). Separate from these two
major functions is their possible role in the control of other
enzyme systems, such as the Bruton-tyrosine kinase (BTK) (66) or the T cell receptor-specific kinase pathways
(Rehm, A., and H.L. Ploegh, unpublished observation;
32), which are perhaps not linked directly to heptahelical
receptors. The classical cycle starts the heterotrimeric G
proteins in its inactive state as a complex of A major challenge for maintaining the specificity with
which G proteins convey signals from heptahelical receptors arises from the complexity of the component parts of
this signaling system (24, 53). An increasing number of
cloned heptahelical receptors must be integrated into a diverse system of effectors and G protein subunits, where 20 Heptahelical receptors are integral membrane proteins
that are synthesized on membrane-bound ribosomes and
inserted into the ER. Many of these receptors carry N-linked
glycans. They reach their final destination from the ER via
the Golgi-complex, and their proper folding and glycosylation depend on this pathway. In contrast to the biosynthetic
route followed by the receptors, the subunits of heterotrimeric G proteins are synthesized on ribosomes in the cytosol.
None of the G protein subunits contain obvious sequences
that would target the newly synthesized polypeptides to
the ER. Modifications such as myristoylation or palmitoylation of the What has been established is the occurrence of heterotrimeric G proteins in specialized membrane domains that
have been referred to as caveolae (38). These specializations are found in the TGN and at the plasma membrane,
are enriched in glycosyl-phosphatidylinositol (GPI)-anchored
membrane proteins, and are relatively resistant to extraction by non-ionic detergents (1, 50). In this study we provide insight into the kinetics of integration of Much of the effort to characterize the specificity of G
protein-receptor interactions has been devoted towards
identifying functional combinations of subunits in transfected cells or in cells manipulated by antisense oligonucleotide injections (29, 51). However, differential targeting
to membrane subdomains may also contribute to the specificity of receptor, G protein, and effector interactions (47).
As a first step towards understanding the assembly of G
protein-coupled signal transduction chains, we here study
the formation of the Antibodies
The Gel Electrophoresis
SDS-gel electrophoresis was performed as described (31), either on 12.5%
or 15% acrylamide gels. Radioactively labeled samples were visualized by
fluorography using DMSO-PPO and exposure to Kodak XAR-5 films.
Immunoblotting
Polypeptides were resolved on gel as indicated in the figure legends and
blotted to nitrocellulose (0.45 µm pore size, Bio-Rad Labs, Hercules, CA).
The blots were incubated with the first antibody, followed by HRF-coupled goat anti-mouse or -goat anti-rabbit immunoglobulin (Southern Biotechnology, Birmingham, AL) antibody. Bound antibody was visualized by
chemoluminescence (ECL detection kit, Kirkegaard and Perry, Gaithersburg, MD) and exposure to Kodak XAR-5 films.
Cell Culture, Metabolic Labeling,
and Immunoprecipitations
The human neuroblastoma cell line IMR-32 was routinely grown in DME
medium, supplemented with 10% FCS, L-glutamine (2 mM), penicillin (1:
1,000 dilution U/ml), and streptomycin (100 µg/ml).
Adherently growing cells were grown to subconfluency, incubated for
45 min in methionine- and cysteine-free DME medium, and then pulselabeled with [35S]methionine/cysteine (80/20) (Express protein labeling
mix, Du Pont-New England Nuclear, Boston, MA) as indicated in the figure legends. For overnight labeling, 50-75 µCi were added to a 10-cm-diam Petri dish of adherently growing cells or to 107 cells in suspension. In
pulse-chase experiments, incorporation of label was stopped by addition
of complete medium supplemented with nonradioactive methionine/
cysteine (1 mM). Brefeldin A (Sigma Chem. Co., St. Louis, MO) was added
at a concentration of 10 µg/ml 2 min before pulse-labeling, and it was
present in the chase medium as indicated in the figure legends. Interferon- For certain experiments, cells were maintained in the absence of FCS
and instead cultured in DME medium supplemented with 0.4% (wt/vol)
bovine serum albumin (Boehringer Mannheim, Indianapolis, IN) for 16-
18 h. Control cells were maintained in DME supplemented with 20% FCS.
Cells were lysed in 1.5 ml NP-40/Lubrol (50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 0.5% NP-40, 0.1% Lubrol, 1 mM EDTA, 1 mM PMSF) lysis
buffer. After 45 min at 4°C lysates were depleted of nuclei and cell debris
by centrifugation for 10 min at 14,000 rpm in an Eppendorf centrifuge,
and then precleared twice with normal rabbit serum and formalin-fixed
Staphylococcus aureus bacteria (Staph. A). Immunoprecipitations for recovery of Endoglycosidase H (Endo H) (New England Biolabs, Beverly, MA) digestion was done as described (68).
Isolation of Detergent-resistant Membrane Domains
In some experiments cells were solubilized in NP-40/Lubrol, and the lysates were centrifuged at 14,000 rpm for 10 min in an Eppendorf centrifuge at 4°C for 10 min. The pellets, corresponding to the detergent-resistant membrane fraction, were solubilized in 200 µl PBS/1% SDS, heated
for 3 min at 95°C, and passed through a 21-gauge needle several times
(59). This procedure was repeated once, and for immunoprecipitation the
solubilized pellets were brought to the same SDS concentration as the NP40/Lubrol supernatants by addition of non-ionic detergent buffer.
To study quantitative changes of detergent-resistant G protein subunit
pools, cells were essentially extracted as described (37). Briefly, cells were
lysed for 30 min on ice with 1.5 ml 25 mM Pipes, pH 6.5, 0.15 M NaCl, 1%
Triton X-100 (Surfact-Amps X-100, Pierce, Rockford, IL). Direct lysis or
re-extraction in the above buffers was done in the presence of 60 mM octyl-glucoside (Calbiochem, La Jolla, CA) (see figure legends).
To purify detergent-resistant membrane domains a modification of a
method described previously was employed (38). Pulse-labeled and -chased
IMR-32 cells were homogenized in 25 mM Pipes, pH 6.5, 0.15 M NaCl,
1% Triton X-100 containing 1 mM PMSF with 20 strokes of a tight-fitting
Dounce homogenizer. The amount of radioactivity was normalized for individual chase points after TCA precipitation of extract aliquots and determination of counts in a Detection of Endogenous GPI-anchored Proteins in
Detergent-resistant Membrane Domains
Detergent-resistant membranes were first raised using the Triton X-100
extraction procedure described before. The resulting detergent-resistant
membrane pellet was then re-extracted for 20 min at 37°C in 1 ml TNE/
Triton X-114 (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,
1% precondensed Triton X-114 [Sigma], 1 mM PMSF, 1 mM leupeptin, 2 µg/ml aprotinin). Membrane extracts were centrifuged at 14,000 rpm for
10 min at 4°C, followed by temperature-induced phase separation of the
supernatant exactly as described (6, 35). Detergent phases were re-extracted three times and then incubated in the absence and presence of phosphatidylinositol-specific phospholipase C (5 U/ml) (PI-PLC; ICN Pharmaceuticals, Costa Mesa, CA) for 1 h at 37°C. After three additional phase separations resulting aqueous phases were quantitatively precipitated by using
acetone. Precipitates were solubilized in Laemmli sample buffer and analyzed on SDS-PAGE.
Subcellular Fractionation
Homogenization.
Cells (suspended in 1 or 2 ml ice-cold homogenization
buffer, 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM
PMSF) were homogenized on ice with 35 strokes of a Dounce homogenizer with a tight-fitting pestle.
Differential Centrifugation.
A postnuclear supernatant was centrifuged
at 100,000 g for 1 h at 4°C to separate cytosol from particulate fractions. A
different protocol was used to further separate plasma membranes from
microsomal membranes (48). Briefly, each homogenate was centrifuged at 12,000 g for 15 min, followed by resuspension of the pellet (plasma membranes, mitochondria), and centrifuged at 28,000 g for 15 min. This additional centrifugation step allowed further removal of cytosolic components and yielded a pellet designated as plasma membranes in this study.
The 12,000 g supernatant of the first centrifugation was centrifuged at
100,000 g for 1 h. The resulting pellet was considered as the microsomal
fraction. Previous studies have shown that the 28,000 g pellet was enriched
for plasma membrane markers, whereas the 100,000 g pellet was enriched
for microsomal membrane enzyme activity (14, 48, 49). All fractions were then lysed in NP-40/Lubrol lysis buffer, followed by centrifugation for 10 min at 14,000 rpm in an Eppendorf centrifuge. The resulting supernatants
were precleared and then immunoprecipitated with ARC9 in the presence
of 0.2% SDS.
Discontinuous Sucrose Gradient.
Homogenates were separated on a sucrose step gradient with minor modifications of a protocol described (7).
A postnuclear supernatant was adjusted to 40% (wt/vol) sucrose in a
buffer containing 10 mM Tris-HCl, pH 7.4, and layered on a 50% (wt/vol)
sucrose solution. Subsequently, a discontinuous sucrose gradient consisting of 0, 15, 25, and 35% (wt/vol) sucrose was poured on top of the 40%
sucrose fraction. The flotation gradient was centrifuged in a SW 41 rotor
at 4°C either at 85,000 g or at 170,000 g for 16 h. 0.5-ml fractions were
taken from the top and an equal volume of 2× lysis buffer was added.
Fractions were either analyzed directly or stored frozen at Tryptic Cleavage Assay
IMR-32 cells were labeled and lysed in 1 ml 50 mM Tris-HCl, pH 7.5, 2 mM
MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.3% (vol/vol) Lubrol for 30 min at
4°C. Lysates were depleted of nuclei and debris, and the soluble supernatant was diluted to 0.075% (vol/vol) Lubrol using the same Tris-buffer
without detergent. Lysates were treated with L-1-tosylamido-2-phenylchloromethyl ketone-treated trypsin (Sigma Chem. Co.) at 30°C for 30 min at the concentrations indicated in the figure legends. Reactions were
stopped by the addition of trypsin inhibitor (Sigma) usually in a 10-fold
excess (wt/wt) over trypsin. Lysates were further supplemented with 1%
(vol/vol) Triton X-100 and 1 mM PMSF before immunoprecipitation.
This assay was also applied to the detection of Kinetics of Formation of Biosynthetic G In pulse-chase experiments using the neuroblastoma cell
line IMR-32, we took advantage of the observation that
the presence of Pulse-chased IMR-32 cells were lysed in the non-ionic
detergents NP-40/Lubrol and equal aliquots of lysate were
immunoprecipitated in the absence (
We then sought to define more accurately when the loss
of immunoreactivity of We conclude that the process of Formation of We verified We establish the occurrence of both characteristic fragments after tryptic digest of IMR-32 cell lysates using mAb
ARC9 and the polyclonal rabbit anti-
In a pulse-chase experiment we observed, using ARC9,
the appearance of this characteristic 27-kD band as coincident with the loss of the SDS-independent form of A similar pattern of digestion products was observed for
Localization of Newly Synthesized G Pulse-chase experiments in combination with subcellular
fractionation were carried out to further resolve
In summary, three different pools of To further resolve the intracellular domains into plasma
membranes, microsomes, and cytosol, a postnuclear supernatant of pulse-chased IMR-32 cells was subjected to differential centrifugation (48). All immunoprecipitations were
done in the presence of 0.2% SDS (Fig. 3 B). The Distribution of G The localization of G protein subunits by immunoblot on
subcellular fractions does not necessarily reflect the processes or kinetics underlying the biogenesis of membrane
domains in vivo. We therefore used pulse-chase labeling in
conjunction with subcellular fractionation on discontinuous sucrose gradients and G protein subunit-specific immunoprecipitation to obtain a kinetic description of transport of G protein subunits in living cells (21).
A homogenate prepared from pulse-labeled IMR-32 cells
was loaded on a sucrose step gradient and centrifuged at
85,000 g for 16 h. Fractions were analyzed individually by
immunoprecipitation with a mixture of anti-G
G Protein Subunits Rapidly Associate with
Detergent-resistant Membranes
When non-ionic detergent insoluble material from labeled
IMR-32 cells was analyzed for the presence of
We next characterized some of the attributes of these
detergent-resistant membranes, as follows. Because the
neuroblastoma cell line IMR-32 used in this study does not
express any of the known isoforms of caveolin (Rehm, A.,
and H.L. Ploegh, unpublished observations), this marker
could not be used to ascertain the presence of proteins
characteristically enriched in the detergent-insoluble domains. Instead, we employed a procedure in which the
TX-100-resistant membrane fraction was re-extracted with
TX-114, and subjected to several rounds of temperatureinduced phase separation (6, 35). This procedure results in
an enrichment of GPI-anchored proteins in the detergent
phase, from which they can be released by treatment with
PI-PLC. The released fraction, recovered in the aqueous
phase after another three cycles of phase separation, was
collected by acetone precipitation and displayed by SDSPAGE (Fig. 5 D). A comparison of the profile of polypeptides released specifically by PI-PLC is indicative of the
presence of GPI-anchored proteins in this detergent-resistant membrane fraction.
As was shown for The presence of
The ratios between soluble and detergent-resistant, membrane-associated
To test for the transport pathway taken by cytosolderived
The kinetics of formation of the G protein Purified recombinant Our experiments did not resolve the location or timing
of the isoprenoid modification of the While the timing and localization of These data suggest that cytosolically synthesized In general, detergent-resistant membranes are operationally defined by their insolubility in non-ionic detergents and by their low buoyant density in sucrose density
gradients (9). They are enriched in GPI-anchored proteins, glycosphingolipids, and cholesterol (17, 40). Detergent-resistant membranes were proposed to play a role in
the sorting of GPI-anchored proteins and viral proteins
destined for the apical surface (57, 58). Segregation into
these glycosphingolipid-enriched domains occurs in the
trans-Golgi network of epithelial cells before transport to
the plasma membrane (9). Other studies have suggested
that detergent-resistant membranes serve other functions
in addition to sorting. These include their role in signal transduction, indicated by the presence of signaling molecules
like the heterotrimeric G proteins, Src family tyrosine kinases, and H-Ras (36, 38). Furthermore, these formations
were shown to have a role in potocytosis, demonstrated by
the internalization of folate (54, 60).
We observe an enrichment of We show that G protein Notwithstanding the occurrence of these detergentresistant membranes, it is likely that proteins associated
with them may display different degrees of detergent resistance. Obviously, the existence of membrane domains refractory to extraction with non-ionic detergents does not
imply that all proteins that reside there are completely resistant to extraction. This could lead to selective loss, upon
detergent extraction, of certain polypeptides from these
domains. G This result seems to be contradictory to a previous report showing that G The topology of luminal receptors and cytosolic G protein subunits adds complexity to the problem of how G
proteins, receptors, and effectors assemble. The timing
and subcellular localization of assembly events may influence the composition of a signaling complex, and hence
determine its specificity. Further analysis of heterotrimeric
G protein assembly would therefore require the integration of a G protein-coupled receptor and downstream effectors in our experimental system.
,
, and a
subunit in which the guanine nucleotide binding site of the
subunit is occupied by GDP (8). Upon receptor activation by an extracellular signal and interaction of the triggered receptors with G proteins, the
subunit releases GDP in exchange for GTP. The trimer then dissociates
into
and
subunits that can now interact independently with effectors (46). The
and
subunits are not
obligatorily derived from this activity cycle, but may also
exist as preformed solitary pools (3, 4, 15).
, 5
, and 8
subunits have been described so far (42, 46).
Even if only a minor fraction of the full combinatorial
complexity of receptors, G protein subunits and effectors
were indeed available, how are the specificity of these interactions and specificity of signaling maintained?
subunits and the prenylation of the
subunits, which provides the
complex with a membrane anchor, occur post- or cotranslationally, presumably in a
cytosolic compartment (10, 39, 41). After G protein activation, myristoylation of certain G protein
subunits may be
required for continued association of
with the plasma
membrane (26, 27, 44). Acylation of the
subunit may further contribute to its affinity for
(34), and the
subunits themselves promote the association of
subunits with phospholipid vesicles in vitro (63). G protein synthesis clearly does not involve lumenal modifications, and the
orientation of G proteins in the final complex with a receptor is cytosolic. Where and when do G protein subunits
assemble with their signaling partners? The oligomerization of G proteins, receptors, and effectors comprises the
assembly of proteins with a lumenal component, as well as
the assembly of the multimeric G protein on the cytosolic face of membranes with the former. To our knowledge,
these issues have not been addressed in living cells.
into detergent-resistant membranes.
complex. To trace biosynthetic intermediates in the assembly of G proteins in living cells,
we made use of anti-
monoclonal antibodies and monoclonal antibodies against
subunits capable of distinguishing between free and assembled
subunits. Through combination of immunoprecipitation, subcellular fractionation,
and pulse-chase analysis we define kinetically distinct
pools of
and
subunits.
Materials and Methods
subunit specific monoclonal antibodies, ARC5 and ARC9, as well
as the polyclonal rabbit anti-
serum, were raised as described (52). The
anti-
o and -
i specific monoclonal antibodies 3C2 and 3E7 have been described (67, 69). The polyclonal rabbit anti-
1 antibody U-49 was prepared
as previously described (43) (kindly provided by Dr. S. Mumby, Dallas,
TX). The Na+/K+-ATPase,
subunit specific antibody served as plasma
membrane marker (gift of Dr. W.J. Nelson, Stanford University, Stanford,
CA). W6/32.HL (anti-MHC class I) has been described extensively (2).
(IFN-
) was added to cell cultures at 50 U/ml for 48 h, where indicated.
subunits were done with 4-5-µl ascites of ARC5 or ARC9
mAb,
subunits were specifically precipitated with either mAbs 3C2 or
3E7, or a mixture of both mAbs (50 µl culture supernatant each). SDS was
added as indicated in the figure legends. Immune complexes were precipitated by adsorption to Staph. A, followed by 4-5 washes in NET buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40).
counter. The homogenate was adjusted to
40% (wt/vol) sucrose and placed at the bottom of an SW-41 centrifuge
tube, and then overlaid with successive layers of 35, 25, 15, and 5% sucrose. After centrifugation at 39,000 rpm for 16 h in a SW41 rotor (Beckman Instruments, Palo Alto, CA), 1-ml fractions were collected from the
top and immediately lysed in 1 ml 2× NP-40/Lubrol lysis buffer supplemented with 0.4% (wt/vol) SDS and 1 mM PMSF.
70°C. The distribution of radioactivity was determined by liquid spectrometry after
TCA precipitation.
complexes in detergent-resistant membrane domains. Briefly, IMR-32 cells were first lysed in
TX-100 containing lysis buffer and then centrifuged at 14,000 rpm for 10 min at 4°C. The resulting pellets were re-extracted with 50 mM Tris-HCl,
pH 7.5, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, and 60 mM octylglucoside. After centrifugation the resulting supernatant was diluted threefold using the same Tris-buffer in the absence of detergent. Trypsin digestion was done as described above.
Results
Intermediates
in a complex with
prevents recognition
by the
-specific mAbs, ARC5 and ARC9 (52). In this
case, the loss of reactivity of
subunits with ARC5 and
ARC9 can be used to monitor assembly of the
complex.
) or presence (+) of
0.2% SDS (Fig. 1 C). At 0 min of chase, the recovery of
from lysates prepared with and without SDS was identical,
whereas at 2.5 min and later time points, we observed a
progressive loss of immunoreactivity from lysates not exposed to SDS. After 30 min essentially no
subunits were detected in the absence of SDS (see also Fig. 1 A). Over
the chase period examined, there is no loss of
in extracts
prepared in the presence of SDS.
Fig. 1.
Detection of biosynthetic intermediates, half-life, and
complex formation of G subunits. (A) IMR-32 cells were pulselabeled for 2.5 min with 150 µCi [35S]methionine and chased for
up to 20 h.
subunits were recovered with mAb ARC9 from nonionic detergent lysates, either in the presence (+) or absence (
)
of 0.2% SDS. Samples in A-C were subjected to 12.5% SDSPAGE. (B) Pulse-labeling of IMR-32 cells for 1.5 min with 150 µCi [35S]methionine (see A); chases were done for the designated
intervals. Precipitations with ARC9 were performed as in A. (C)
IMR-32 cells were pulse-labeled for 5 min with 150 µCi [35S]methionine and chased for the times indicated.
subunits were immunoprecipitated with mAb ARC9 in the absence (
) or presence (+) of 0.2% SDS.
[View Larger Version of this Image (55K GIF file)]
with ARC9 starts. In a pulsechase experiment where we limited the pulse to 1.5 min
(Fig. 1 B), equal amounts of
were again obtained at 0 min chase for lysates prepared plus and minus SDS. At
2.5 min chase, we observed a loss of the minus SDS form,
with a further reduction at 10 min chase. Despite the fact
that equal numbers of cells were used at each chase point, we consistently observed that the total amount of radioactivity recovered was maximal between 5 and 7.5 min of
pulse or chase (note that the use of equal numbers of cells
instead of normalized amounts of radioactivity per chase
point allows the detection of these differences in recovery). This observation may reflect differences in detergent
extractibility of
subunits due to the lipid environment in
which the
subunits find themselves. While
subunits
move through the cell, this lipid environment is likely to
change (see below).
complex formation
is a rapid event starting within the first 2.5 min after completion of the polypeptide chains. The half-life of the
subunit in IMR-32 cells labeled for 2.5 min and chased for
20 h (Fig. 1 A) was in excess of 20 h.
Complexes Determined by
Tryptic Proteolysis
complex formation in a pulse-chase experiment using a trypsin-protection assay. Upon association
with
, the
subunit is partially protected from proteolysis
by trypsin, and gives rise to a 27-kD COOH-terminal fragment and a 14-kD NH2-terminal fragment, as it has been
shown for the combination
1
2 (16, 18, 64).
serum (Fig. 2 A).
Although we tested different concentration ranges of trypsin, we observed in these experiments the persistence of
full-length forms of
, indicating that different subtype
combinations of
and
in IMR-32 cells may exist with
different degrees of trypsin susceptibility, or that at least
some fraction of
may not be fully accessible to trypsin,
unlike purified
subunits. Re-immunoprecipitation of
the 27-kD fragment with the peptide specific polyclonal
antibody U-49 (derived against amino acids 131-145 of
1)
established the origin of this fragment as
subunit-derived
(Fig. 2 B).
Fig. 2.
The generation of the subunit derived 27-kD tryptic
cleavage product in cell lysates is coincident with the appearance of newly assembled
complexes. (A) IMR-32 cells were labeled with 200 µCi [35S]methionine for 2 h. Cells were lysed in 0.3%
Lubrol containing lysis buffer and trypsin was added at the concentrations indicated. Immunoprecipitations were done in the
presence of 0.2% SDS using ARC9 or rabbit anti-
serum. The
positions of undigested
chains and tryptic fragments are indicated on the right. (B) The origin of the 27-kD tryptic
fragment
was confirmed by re-immunoprecipitation of ARC9 immunoprecipitates with the peptide-specific polyclonal antiserum U-49. (C)
IMR-32 cells were pulsed for 2 min with 500 µCi [35S]methionine
and chased for the times indicated. Lysates were prepared as in
A, and trypsin was added at a concentration of 50 µg/ml. Immunoprecipitations were done in the presence and absence of 0.2%
SDS. Note that the appearance of the 27-kD fragment is coincident with the loss of the (
) SDS form in untreated samples. (D)
and
subunits are complexed in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled for 3 h with 150 µCi [35S]methionine and lysed in TX-100-containing lysis buffer.
Detergent-resistant membranes were resolubilized in octyl-glucoside containing lysis buffer. Trypsin digestion was done as described
before (A-C); immunoprecipitations with ARC9 and rabbit anti
serum resulted in the appearance of the 27-kD and 14-kD
tryptic fragments.
[View Larger Versions of these Images (41 + 32 + 22K GIF file)]
, indicating that at this chase time (10 min), association of
and
has occurred (Fig. 2 C). The NH2-terminal derived 14-kD
fragment could be immunoprecipitated with the polyclonal
rabbit anti-
antiserum (data not shown). The 27-kD
fragment, which is devoid of the
subunit after tryptic digestion, can also be immunoprecipitated with ARC9 in the
absence of SDS (Fig. 2 C). The difference in recovery of
this 27-kD fragment compared to immunoprecipitations in
the presence of SDS is due to incomplete inhibition of
trypsin activity in the absence of SDS. Accordingly, we
found substantial amounts of low molecular weight degradation products present at the dye front.
's recovered from non-ionic detergent-insoluble domains (see below), confirming the occurrence of
as a
complex (Fig. 2 D).
Subunits
subunit
intermediates. When we fractionated cells simply into a
particulate or membrane fraction (M) and a cytosolic fraction (C), and immunoprecipitated
in the absence and
presence of SDS, we observed three distinct pools of
subunits (Fig. 3 A). At 0 min chase, the majority of
was
detected in the C fraction as the free, uncomplexed form. After 2.5 min of chase, the ratio between cytosolic and
membrane-associated
was slightly shifted, with
still
found predominantly in the cytosol. Starting at 7.5 min of
chase, the recovery of
in the absence of SDS was significantly reduced. The loss of free
indicates the formation
of complexes that are different from those at 0 and 2.5 min
of chase. At the 15-min chase interval, the distribution between cytosolic (C)- and membrane (M)-associated
appeared to be equivalent, whereas their ratio finally shifted
towards the M fraction after 35 and 60 min of chase. After
the 60-min chase interval, only a minor amount of
remained in the cytosolic compartment.
Fig. 3.
Intracellular redistribution of newly synthesized G subunits. (A) IMR-32 cells were pulse-labeled with 150 µCi [35S]methionine for 2.5 min and chased for the times indicated. A crude homogenate was separated into microsomes and cytosol by centrifugation
at 100,000 g for 1 h at 4°C. The pellets recovered were considered the M fractions and contained plasma membranes and microsomal
membranes, whereas the supernatant contained the cytosol (fraction C). Solubilization of subcellular fractions in NP-40/Lubrol lysis
buffer was as for Fig. 1. G
was recovered in the absence (
) or presence (+) of 0.2% SDS. mAb ARC9 immunoprecipitates were analyzed on a 12.5% SDS-PAGE. (B) IMR-32 cells were pulse-labeled as in A and chased for up to 180 min. Membrane and cytosol fractions were prepared by differential centrifugation, yielding fractions designated plasma membranes (28,000 g pellet); microsomes (100,000 g
pellet); and cytosol (100,000 g supernatant). All immunoprecipitations with ARC9 were done in the presence of 0.2% SDS. Samples
were resolved by 12.5% SDS-PAGE. Although at 15-min chase some of the total cell lysate was inadvertently lost in this experiment,
the ratio between the cytosolic and microsomal
as the relevant parameter can still be evaluated. (C) The plasma membrane marker
-Na+/K+-ATPase is detectable in the 28,000 g membrane fraction, but not in the 100,000 g pellet. 50-µg aliquots of membrane fractions as obtained in B were subjected to electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. The blotting membrane was incubated with rabbit anti-
-Na+/K+-ATPase antiserum.
[View Larger Versions of these Images (23 + 59 + 26K GIF file)]
subunits could be
identified. Pool 1 is defined as a free, uncomplexed state,
and the second pool comprises cytosolic
subunits,
whose contribution to the total pool diminishes with time.
Pool 3 contains membrane-bound
subunits and requires
inclusion of SDS for visualization. This fraction increases
with time and is recruited from the cytosolic pool. It is reasonable to suggest that this membrane-bound fraction is in
a complex with
subunits.
subunit was recovered at 0 min of chase predominantly from
the cytosolic fraction and only a minor amount was associated with lower density microsomal membranes. The maximum amount of
obtained from cytosol or microsomes
was found at 2.5 min of chase, followed by a decrease in
both compartments of up to 45 min. The
subunits appeared after 15 min of chase in the plasma membrane
pool, which contains plasma membranes, as confirmed by
the occurrence of the plasma membrane marker
-Na+/
K+-ATPase (Fig. 3 C), which may also contain high density microsomes. The activity of
recovered from this
compartment strongly increased at the 30-min chase interval. The microsomal and cytosolic pool of
remained at a
constantly low level after 45 min of chase. We conclude
that movement of
is characterized by a decrease of
in
the cytosolic and microsomal fractions and a corresponding increase in the plasma membrane pool.
and G
Subunits Assessed by
Subcellular Fractionation
mAbs (3C2/
3E7) or an anti-G
mAb (ARC9, plus 0.2% SDS). The distribution of both radiolabeled
or
in these gradients
changed with time (Fig. 4, A and B). At the early time of
chase (0 min),
and
were recovered from the denser region of the gradient, reflecting a status where
subunits,
shortly after synthesis, are devoid of
subunits, but they
still fractionate with large dense particles, such as the cytosolic ribosomes on which they are synthesized (7). At the 20-min chase point we noted a movement of
and
subunits towards lower density fractions (B, C, and D interface). This result is consistent with the observation that after 20 min of chase most
subunits are complexed with
subunits, and require SDS for efficient recovery in immunoprecipitation (Figs. 1 and 3). The assembly of
with the
subunit is likely to provide the
complex with a membrane anchor, in the form of the COOH-terminal prenyl modification of the
subunit. This anchor modification
would allow part of the complexes to associate with lower
density membranes, which are found in the B and C interface. A fraction of
subunits was still detectable in the D
and E interface, where both plasma membranes (D interface), large particles and lysosomal membranes, fractionate (E interface) (7). A similar result was obtained for the
subunit, which at the 0-min chase point was found in the
high density region of the gradient and after a 20-min
chase moved to the low density fraction. At 60 min of
chase we observed that G
exhibited a more prominent bimodal distribution as compared to
. The peaks of this
broad distribution were centered around the C and D interface. This result is consistent with the observation that
the C interface accumulates light density membranes with
the buoyant density of Golgi membranes, whereas plasma
membranes characteristically float to the high density region (D interface) of the sucrose gradient (7). At 60 min of
chase we recovered little material from the dense region of
the gradient with the anti-G
mAb ARC9, indicating that
formation of trimers in domains fractionating at the D interface might not be stoichiometric. We performed chases
up to 240 min to reveal progressive changes of G protein
subunit distribution as a function of time (data not shown),
but no obvious alterations in comparison to the 60-min chase interval were discernible. This observation suggests
that movement of G protein subunits from their site of
synthesis to their final destination is essentially complete
at 60 min of chase.
Fig. 4.
Domain localization of newly synthesized G and G
subunits. IMR-32 cells were pulse-labeled for 5 min with 500 µCi
[35S]methionine and chased for the designated times. Cells were
homogenized as shown (Fig. 3) and resolved on a discontinuous
sucrose gradient. 0.5-ml fractions were collected from the top of
each gradient, lysed in an equal volume of 2× NP-40/Lubrol lysis
buffer, and aliquots from each fraction were immunoprecipitated
with ARC9 (+ 0.2% SDS) (A) or with a mixture of anti-G
antibodies (3E7, 3C2) (B). The interfaces between different sucrose
densities are designated A-E, whereas the sucrose density steps
are represented in percent sucrose. The distribution of radioactivity along the gradients is shown in C. Data shown are representative for at least three experiments performed in duplicate.
[View Larger Versions of these Images (43 + 46 + 18K GIF file)]
subunits,
we found substantial amounts of
subunits even after two
rounds of non-ionic detergent extraction, although the majority of
subunits was still recovered from the detergent
soluble fraction (Fig. 5, A and B). Whereas the recovery of
from the soluble extract required SDS, the efficiency of
recovery was independent of SDS, an indication that the
epitopes recognized on
are freely accessible. To determine the time point when
associates with the insoluble
pellet, IMR-32 cells were pulse-labeled for 2.5 min, chased
for the times indicated, and extracted with NP-40/Lubrol.
The
subunit was found in the pellet beginning at 1 min of
chase (Fig. 5 C).
Fig. 5.
G and G
are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100 µCi of [35S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C
for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from
the supernatant were done either in the presence (+) or absence (
) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover
subunits and
was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A-C). Arrows indicate the positions of
and
subunits. (B) IMR-32 cells were labeled for 2 h with 250 µCi [35S]methionine followed by solubilization in NP-40/ Lubrol lysis buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of
G
subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 µCi [35S]methionine and
chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was
re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic
detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 µCi [35S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114-containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (
) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins.
Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows
on the right indicate the appearance of some of the proteins specifically released by PI-PLC.
[View Larger Versions of these Images (46 + 23K GIF file)]
complexes in soluble detergent extracts (Fig. 2, A-C), we established that
complexes
present in the detergent-resistant membranes yielded a
pattern of tryptic products consistent with their proper assembly (Fig. 2 D).
complexes and other proteins involved in signal transduction in detergent-resistant domains has been attributed physiological significance. It has
been argued that these detergent-resistant membranes fulfill a specialized role in signaling by allowing a high local
concentration of the relevant proteins. We sought to explore this issue by comparing the relative amounts of
in
detergent-resistant membranes from cells held under serum starvation vs cells maintained in 20% FCS. For cells
cultured in the presence of FCS, we observed that ~30%
of all
subunits could be retrieved in the detergent-resistant membranes, where it must be kept in mind that even
non-ionic detergent may extract some
's (and other
membrane proteins) from areas generally defined as detergent-resistant membranes (Fig. 6 A). When
content
was analyzed for cells maintained in the presence or absence of serum (normalized for the amount of protein
loaded for each sample, Fig. 6 B), we observed an approximately threefold enrichment of
's in detergent-resistant
membranes from serum-stimulated cells.
Fig. 6.
complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 106) were lysed directly in
PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells
was first extracted with TX-100-containing lysis buffer, followed
by re-extraction of the detergent-resistant membrane fraction in
PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100-resistant
pellet were heated twice for 3 min at 100°C in the presence of
SDS, and passed through a 21-gauge needle several times. Laemmli
sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein
subunits. (B) Equal numbers of cells were cultured for 16-18 h in the presence (+) or absence (
) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described
in A. Samples were normalized for the amount of protein (100 µg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-
mAb ARC5.
[View Larger Versions of these Images (41 + 22K GIF file)]
subunits were examined in a pulsechase experiment using TX-100-containing lysis buffer.
Detergent-resistant pellets were then resolubilized either
in 1% SDS (see above) or octylglucoside; the latter was
found to solubilize
and
subunits with an efficiency comparable to SDS (Rehm, A., and H.L. Ploegh, unpublished observations). The detergent-resistant pool of
increased until 30 min, when a plateau was reached (Fig. 7,
A and B). Further evidence for a kinetic shift in association of
with detergent-resistant domains is shown in Fig.
7 C, where the amount of low density protein-lipid complexes, separated by sucrose density gradient centrifugation, increased after 40 min of chase.
Fig. 7.
Quantitative changes of the Triton-insoluble subunit
pool shortly after synthesis. (A) IMR-32 cells were pulsed with
500 µCi [35S]methionine and chased for the times indicated. Cells
were lysed in TX-100 lysis buffer and separated as described in
Fig. 5. The pellet was resolubilized in 60 mM octylglucoside, and
the amounts of radioactivity for individual chase points were normalized for the soluble and pellet fraction. Immunoprecipitations
were done in the presence of 0.2% SDS using mAb ARC9. (B)
IMR-32 cells were pulsed and chased as indicated. Extractions
and immunoprecipitations were done as described in A. (C)
IMR-32 cells were pulsed for 1.5 min with 500 µCi [35S]methionine and chased for 5 or 40 min. Cells were homogenized in TX100 lysis buffer, incorporation of radioactivity was determined, and extracts containing equal amounts of counts were resolved on a discontinuous sucrose gradient. 1-ml fractions were collected from the top, lysed in an equal volume of 2× NP-40/Lubrol lysis buffer supplemented with 0.4% SDS. Immunoprecipitations were
performed with ARC9.
[View Larger Version of this Image (49K GIF file)]
subunits to rapidly formed detergent-resistant
domains, ER to Golgi transport was blocked by BFA. We
scored for inhibition of intracellular glycoprotein transport by analyzing persistence of sensitivity of MHC class I
molecules to Endo H (Fig. 8, B and C). The association of
subunits with detergent-resistant membranes was not
affected by the BFA-imposed block of ER to Golgi transfer (Fig. 8 A). We conclude that cytosolic
subunits are
inserted into detergent-resistant membranes at a site distal
from the BFA block, probably at the plasma membrane itself.
Fig. 8.
Transport and formation of detergent-resistant membranes is insensitive to BFA treatment. IMR-32 cells were stimulated for 48 h with 50 U/ml IFN-. Cells were pulsed in the presence and absence of 10 µg/ml brefeldin A for 10 min with 500 µCi
[35S]methionine and chased (+ and
10 µg/ml BFA) for the time
indicated. Cells were extracted as described in Fig. 7 and separated into a soluble and a TX-100-resistant fraction. Resolubilization of the pellet was done with octylglucoside containing lysis
buffer. ARC9 was used to recover
subunits (+0.2% SDS) (A),
and MHC class I heavy chains were immunoprecipitated with
W6/32.HL (B and C). Endo H digestions of W6/32.HL precipitates were done to demonstrate the effects of anterograde ER to
Golgi transport block. BFA renders MHC class I molecules endo
H sensitive even after 60 min of chase.
[View Larger Version of this Image (56K GIF file)]
Discussion
complex,
and the kinetics with which these
subunits are targeted
to distinct membrane domains were the subject of this study.
Newly synthesized
and
subunits are detectable by immunoprecipitation within 1.5 min of labeling, and their halflife is in excess of 20 h (Fig. 1) (for
, see reference 33).
Anti-
monoclonal antibodies, raised against immunoaffinity purified
, provided the tools with which to follow
biosynthetic intermediates of
subunits. Formation of the
complex leads to masking of the epitope recognized by
these mAbs on the
subunit by the association of the
subunit (52). Coincident with this loss of immunoreactivity, we observe, upon proteolysis with trypsin, the appearance of a characteristic fragment seen only for properly assembled
subunits (Fig. 2) (16, 18, 64). Consequently,
the pattern of immunoreactivity, seen with the anti-
mAbs, could be used to follow
assembly in living cells.
and
subunits, in vitro translated
subunits or
subunits transfected into cells have
been used to explore the mechanisms and structural elements required for
subunit assembly (10, 45, 70). Prenylation of
is not required for
assembly, but this modification of the
subunit apparently facilitates the interaction
of preformed
complexes with the
subunit (25). The
assembly of
apparently occurs before removal of the 3 carboxy-terminal amino acids from the prenylated
subunit (70). The enzymatic reactivity required for this reaction is largely localized to the microsomal membranes (22,
23). Therefore, the assembly of
and prenylation are predominantly cytosolic processes that likely precede membrane attachment. Prevention of
prenylation in living cells,
either by site-directed mutagenesis of the prenylation target sequence or by inhibition of isoprenoid precursor synthesis, inhibits membrane targeting of the
complex. It is
not known whether the isoprenoid moiety interacts with
the lipid bilayer directly or facilitates interaction with the
membrane by means of lipid-adaptor-protein interaction
(13). Our data are consistent with
complex formation starting in the cytosol, although the process may continue
for
or
subunits that are already membrane-associated.
subunit. The farnesyl moiety of the Ras protein facilitates targeting of the protein to detergent-resistant membranes in living cells, whereas
a nonfarnesylated mutant of Ras fails to associate with this
domain (62). In support of the role of the isoprenoid anchor in targeting to detergent-resistant membranes, prenylation of G
in yeast was shown to be a requirement for
this membrane association (30).
modification
and assembly has been addressed, kinetics of assembly on
differential membrane subdomains have not been established. Our experiments have thus far failed to identify the
intracellular site at which
subunits associate with the
complex. As far as the formation of the
complex is concerned, we note that its association with the detergentresistant membrane fraction is rapid and appears not to be
affected by BFA treatment (Fig. 8 A). One of the most pronounced effects of BFA is the tight block of membrane
traffic out of the ER, whereas the retrograde movement of
Golgi membrane components into the ER continues (28).
We conclude that a sizable fraction of G protein subunits,
associated with these insoluble domains, probably do not
associate with the heptahelical receptors at the level of the
ER but do so later. The latter presumably depend on the
anterograde pathway through ER and Golgi for proper folding and maturation of their N-linked glycans.
subunits, once formed, can be targeted directly to the plasma
membrane. Alternatively, the BFA treatment of cells could
lead to a redistribution of Golgi-membrane derived lipids,
causing a detergent resistance of proteins which associate
with them. We consider this possibility unlikely since the
induction of an enhanced retrograde trafficking pathway
by BFA from the Golgi to the ER does not include the redistribution of components of the trans-Golgi network,
where the occurrence of detergent-insoluble domains has
been reported (28). Second, it is possible that other intracellular membranes, including the ER, may contain such
detergent-insoluble domains. Insolubility may not necessarily be caused by the occurrence of
subunits in association with glycolipid rafts, which are expected to be largely
absent from the ER (see below). A third possibility is suggested by the observation that proteins might actively recruit lipids to create detergent-insoluble domains. One example is the coclustering of a lipid into detergent-resistant
membranes following aggregation of the IgE receptor
(65). In this view, the newly assembled cytosol-derived
subunits, upon attachment to a membrane, may recruit lipids that render the
subunits resistant to solubilization with non-ionic detergents. The alternative interpretation
would involve the creation of, and recruitment of,
subunits to a detergent-resistant membrane fraction in the ER
itself. This possibility must be considered unlikely if such
detergent resistance is caused largely by incorporation of
glycolipids.
's in detergent-resistant
membranes when comparing serum-exposed vs serumstarved cells. This observation might relate to the cholesterol depletion that follows serum starvation, because cholesterol renders membrane domains more resistant to
extraction with non-ionic detergents. This result suggests
that the formation of detergent-resistant membranes, and
hence the proteins found herein, is controlled by the lipid phase of these domains (11, 61).
and
subunits rapidly associate with detergent-insoluble membranes. When cellular extracts were separated into a non-ionic detergent-soluble
fraction and an insoluble pellet, substantial amounts of
and
subunits were recovered in association with the insoluble pellet already after 1 min of chase (Fig. 5 C). Transient appearance in detergent-resistant membranes has
also been described for human placental alkaline phosphatase and for influenza virus hemagglutinin (HA), albeit with different kinetics of association (9, 59). HA acquires some detergent insolubility, but the association of HA
with the insoluble pellet is transient and found only after
15-20 min of chase. This fraction of HA was localized to
the trans-Golgi network and plasma membrane. We found
that
subunits associated with detergent-resistant membranes can undergo ADP-ribosylation (Rehm, A., M. Yilla,
and H.L. Ploegh, unpublished observation), indicating that at least transient interactions between
and
subunits in this compartment must occur. The recovery of
from detergent resistant membranes after resolubilization
in octylglucoside required the presence of SDS, demonstrating that
and
are complexed in this domain. Furthermore, the tryptic digestion of detergent-resistant
results in 27- and 14-kD fragments, supporting our previous notion that
and
find themselves associated in this domain (Fig. 2 D).
's are known to undergo rapid cycles of acylation and deacylation in the course of receptor activation (55), whereas the isoprenoid moiety on the
subunit is
probably more stable. Our observation that
's but not
's remain associated with the detergent-resistant membranes after a second extraction step may be related to the
different properties and dynamics of the lipid modifications of G protein subunits.
subunits but not
complexes remain detergent resistant after TX-100 extraction. We conclude that the different protocols applied to enrich for
detergent-resistant membranes might account for the differences observed. Although we have analyzed a complex
cellular lysate after extraction with TX-100, Chang et al.
(12) have enriched detergent-resistant caveolae with a detergent-free method and then treated those membranes with a non-ionic detergent. Therefore, it is likely that an altered protein-detergent ratio has influenced the quantitative recovery of
complexes from detergent-resistant domains.
In addition, our data are supported by recent reports in
which methods were applied for the purification of detergent-resistant membranes that are similar to the one used
here (20, 62). Based on the purification criteria presented
in our study we consider it unlikely that all
complexes
are in the detergent-resistant membrane domain and are
only partially removed by TX-100 extraction. Homogenization of cells in the absence of detergent results in kinetically and spatially distinguishable cytosolic, plasma membrane and microsomal pools; the latter contain endosomal
and lysosomal membranes not reported to exhibit detergent
resistant properties. Second, the kinetics of acquisition of
detergent resistance indicate that even if this domain would
constitute the final destination for
complexes, there
still exist pools of soluble
complexes that could be intermediates, as revealed by immunoprecipitation and pulsechase analysis.
Received for publication 24 September 1996 and in revised form 8 January 1997.
1. Abbreviations used in this paper: Endo H, endoglycosidase H; G proteins, heterotrimeric guanine nucleotide-binding proteins; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; IFN-We are grateful to Drs. Mamadi Yilla and Uta Höpken for review of the manuscript. We also thank Hisse Martien van Santen, Robert Machold, Johannes Huppa, and Matthew Bogyo for their critical evaluation of the manuscript.