From the Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, December 27, 2002, and in revised form, February 8, 2003
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ABSTRACT |
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Nascent Heterotrimeric G
proteins1 are composed of Mechanisms underlying the PM targeting of the Previously we found that the Cell Culture--
HEK293 and COS7 cells were obtained from the
American Type Culture Collection (Manassas, VA) and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and maintained at 37 °C in a 95% air, 5%
CO2-humidified atmosphere.
Constructs--
Wild type Transfection--
Unless otherwise noted, cells were seeded 1 day before transfection, and 1 µg of total plasmid DNA at a 6:3:1
ratio of Immunofluorescence Microscopy--
Cells were fixed with 3.7%
formaldehyde in phosphate-buffered saline (PBS) for 15 min and
permeabilized by incubation in blocking buffer (2.5% nonfat milk and
1% Triton X-100 in Tris-buffered saline) for 20 min. Cells were then
incubated with the indicated primary antibodies in blocking buffer for
1 h. The cells were washed with blocking buffer and incubated in a
1:250 dilution of a goat anti-mouse or a goat anti-rabbit antibody
conjugated with either Alexa 488 or Alexa 594 for 30 min. The
coverslips were washed with 1% Triton X-100 in Tris-buffered saline,
rinsed in distilled water, and mounted on glass slides with Prolong
Antifade reagent (Molecular Probes, Eugene, OR). Microscopy was
performed with an Olympus BX60 microscope. Images were recorded with a
Sony DKC-5000 digital camera and transferred to Adobe Photoshop for digital processing.
Confocal Microscopy--
Coverslips were prepared for confocal
microscopy as described above under "Immunofluorescence
Microscopy." Representative images were recorded by confocal
microscopy at the Kimmel Cancer Center Bioimaging Facility using a
Bio-Rad MRC-600 laser scanning confocal microscope running CoMos 7.0a
software and interfaced to a Zeiss Axiovert 100 microscope with Zeiss
Plan-Apo 63 × 1.40 NA oil immersion objective. Dual-labeled
samples were analyzed using simultaneous excitation at 488 and 568 nm.
Images of "x-y" sections through the middle of a cell were recorded.
Ni-NTA Pull Down of Prenylation Assay--
COS7 cells were seeded in 60-mm culture
plates. 24 h later the cells were transfected with the indicated
plasmids (Myc-His tagged Cell Fractionation Assay--
Soluble and particulate fractions
were isolated as described previously (10). Briefly, 48 h after
transfection HEK293 cells were washed in ice-cold PBS and lysed in
hypotonic lysis buffer (50 mM Tris-HCl, pH 8, 2.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin). Cells were passed through a 27-gauge needle 10 times. Lysed cells were centrifuged at 400 × g for 5 min to remove nuclei and debris. The supernatant was centrifuged at
150,000 × g for 20 min at 4 °C. Fractions were analyzed by SDS-PAGE and immunoblotting using the indicated antibody.
Materials--
pEYFP-Mito vector (Clontech,
Palo Alto, CA) was provided by Emad Alnemri (Thomas Jefferson
University). pEYFP-IBV-M1 encoding an ER marker protein was a generous
gift from Mark R. Philips (New York University). 9E10 monoclonal
antibody was from Covance (Berkeley, CA). 12CA5 monoclonal antibody was
from Roche Applied Science. Anti-HA and anti- In the present study, we focused on the
and
subunits of heterotrimeric G
proteins need to be targeted to the cytoplasmic face of the plasma
membrane (PM) in order to transmit signals. We show that
1
2 is poorly targeted to the PM and
predominantly localized to endoplasmic reticulum (ER) membranes when
expressed in HEK293 cells, but co-expression of a G protein
subunit allows strong PM localization of the
1
2. Furthermore, C-terminal
isoprenylation of the
subunit is necessary but not sufficient for
PM localization of
1
2. Isoprenylation of
2 and localization of
1
2
to the ER occurs independently of
expression. Efficient PM
localization of
1
2 in the absence of
co-expressed
is observed when a site for palmitoylation, a putative
second membrane targeting signal, is introduced into
2.
When a mutant of
s is targeted to mitochondria,
1
2 follows, consistent with an important
role for
in promoting subcellular localization of
.
Furthermore, we directly demonstrate the requirement for
by showing
that disruption of heterotrimer formation by the introduction of
binding mutations into
1 impedes PM targeting of
1
2. The results indicate that two
membrane targeting signals, lipid modification and
binding, make
concerted contributions to PM localization of
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits. The
complex only dissociates when denatured
and hence is a functional monomer under physiological conditions. Upon
receptor activation the
dimer is freed from GTP-bound
and
relays signals to downstream molecules until it reassociates with
GDP-bound
, re-forming the heterotrimer. To perpetuate this G
protein cycle, the trimer must be tethered to the cytoplasmic face of
the PM. This crucial subcellular localization is promoted by the
covalent attachment of lipids to the subunits. Three lipid
modifications have been found in G proteins, namely myristoylation
and/or palmitoylation for the
subunit and isoprenylation for the
subunit. Myristoylation is the covalent attachment of a 14-carbon
saturated myristate to an N-terminal glycine through an amide bond,
whereas palmitoylation is a 16-carbon saturated palmitate linked to a
cysteine via a thioester bond. Isoprenylation is a lipid modification
in which an unsaturated, 15-carbon farnesyl isoprenoid or 20-carbon
geranylgeranyl isoprenoid is linked to a cysteine, via a thioether bond.
subunit have been
studied in some detail (1-3). The available data suggest a model in
which myristoylation and/or binding to
subunits constitutes an
initial membrane targeting signal for the
subunit. Subsequently,
palmitoylation functions as a second signal that specifies localization
to the PM. In contrast to the
subunit, relatively less is known
about how
is targeted to the PM. Either a 15-carbon farnesyl or
20-carbon geranylgeranyl isoprenoid is linked to a cysteine residue in
the so-called CAAX motif in the C terminus of the
subunit (4, 5). The CAAX box (where C is a cysteine,
A is commonly an aliphatic amino acid, and X can
be one of several amino acids) is a consensus sequence for isoprenylation. The X residue is thought to specify which
isoprenoid group will be linked to the cysteine. Among 12 human
subunits thus far identified,
1,
9, and
11 have serine in the X position and are
farnesylated, and the rest of them have leucine and are modified with a
geranylgeranyl group. It has been generally agreed that isoprenylation
of the
subunit is essential in PM targeting of the dimer but
whether the modification is sufficient has not been defined. Other
prenylated proteins, such as members of the Ras family of small
GTPases, require an additional second signal for their PM targeting (6,
7). H-Ras and N-Ras are palmitoylated at cysteines upstream of the
prenylcysteine, whereas K-Ras contains polybasic lysines adjacent to
the CAAX box. Ste18p, the
subunit in Saccharomyces
cerevisiae, is modified with palmitate at the cysteine immediately
next to the prenylcysteine (8). However, none of the human
have
such cysteines or a stretch of basic residues flanking its
CAAX motif.
complex was localized poorly to the
PM when transiently expressed in HEK293 cells, whereas co-expression of
the
subunit led to strong PM localization of
(9). This
suggests that the complete information required for efficient PM
targeting of
is not contained within the
dimer, and
interaction of the
dimer with the
subunit is critical for PM
targeting of
. In this report, we examined the importance of the
subunit in PM targeting of
. We show that isoprenylation of
the
subunit is necessary but not sufficient for PM localization of
, and expression of
is not required for
isoprenylation. We demonstrate that introduction of an additional membrane targeting signal into the
subunit can overcome the reliance of
on
for PM targeting. In addition,
accompanies an
subunit
mis-targeted to mitochondria. Finally, we present the first direct test
of the necessity for heterotrimer assembly for PM localization of
by demonstrating that
binding-deficient mutants of
fail to localize to the PM, even when co-expressed with
. The
results presented herein are consistent with a model in which both
heterotrimer assembly and lipid modifications, working in concert,
target
and
to the PM.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s (HA-tagged) and
-binding-deficient mutant
sIEK+(HA-tagged) were
described previously (10). The expression vector for
1 (Myc- and His-tagged) was provided by David P. Siderovski (University of North Carolina) (9).
-Binding
defective
1 mutants,
1I80A,
1N88A/K89A,
1L117A,
1D228R,
1D246S,
1N88A/K89A/D246S (hereafter named
1NKD),
1N88A/K89A/D228R/D246S (named
1NKDD), and
1I80A/N88A/K89A/D228R/D246S (named
1INKDD), were created using QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) and Myc-His tagged
1 in pcDNA3.1 as the template. Non-tagged
2 was described previously (9).
2
(Myc-tagged) and
2MITO in pcDNA3 (11) were provided
by Henry R. Bourne (University of California, San Francisco).
2F66C,
2F67C, and
2C68S
were made from
2 (no tag) using the QuickChange kit and
so was Myc-tagged
2C68S. A mitochondrial targeting
sequence was excised from
2MITO and inserted into wild
type
s (HA-tagged) to create
s fused with
mitochondrial targeting sequence (mito-
s). pcDNA3
containing His-tagged
1 was provided by Tohru Kozasa
(University of Illinois, Chicago).
:
:
was transfected into the cells using FuGENE 6 (Roche Applied Science). Cells were incubated overnight, transferred to
new plates, and grown for 24 h prior to subsequent manipulation.
1--
Transfected cells were
washed once with ice-cold PBS and lysed in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 2.5 mM MgCl2) supplemented with protease inhibitors
(1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin). After 1 h of incubation on ice, nuclei and
insoluble material were removed by centrifugation. Ni-NTA magnetic
agarose beads (Qiagen, Valencia, CA) were added to the clarified
lysate, and the samples were tumbled for 2 h at 4 °C. The
samples were washed three times and eluted with elution buffer
containing 250 mM imidazole. Eluates were separated by SDS-PAGE followed by immunoblotting. Bands were visualized by chemiluminescence (Pierce). These experiments utilized the
hexahistidine tag of N-terminal Myc-His-tagged
1.
1 and non-tagged
2 without or with HA-tagged
s). Cells were incubated overnight and labeled with 50 µCi/ml
[3H]mevalolactone (American Radiolabeled Chemical, St.
Louis, MO) for another 18 h in the presence of 10 µM
mevastatin (Biomol, Plymouth Meeting, PA). Cells were washed with
ice-cold PBS and lysed. The
complex was pulled down and purified
using Ni-NTA agarose beads as described above. Eluates were separated
by SDS-PAGE and transferred onto polyvinylidene difluoride membrane.
The membrane was sprayed with EnHance (PerkinElmer Life Sciences) and
then exposed to Hyperfilm MP (Amersham Biosciences) at
80 °C for
8-15 days. After fluorography, the
2 subunit was
detected by immunoblotting. Note that because an anti-
2
polyclonal antibody did not recognize
2C68S, His-tagged
1 (12) and Myc-tagged
2C68S were used for the
s
1
2C68S control
sample, and
2C68S was detected on an immunoblot with an
anti-Myc monoclonal antibody.
2 rabbit
polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s
1
2 G protein
heterotrimer. Immunofluorescence microscopy was utilized to examine the
subcellular localization of the
and
subunits. It has been
shown that
s is predominantly localized to the PM when
expressed alone (9, 10). As described by us previously (10), when
1 and
2 were expressed together in HEK293
cells very little PM localization was observed (Fig.
1, a and b).
However, when
s was co-expressed with
1
and
2,
s and
1 strongly
co-localized at the PM (Fig. 1, c and d).
Expression of
q also strongly promoted PM localization
of
1
2 (not shown).
2
displayed PM localization when
s,
1, and
2 were all expressed together, but
2 was
also found intracellularly and not co-localizing with
s
or
1 (Fig. 1, e-h). Apparently some of the
2 did not form a dimer with
1. Because
localization of
1 rather than
2 appeared
to be a better representative of the
1
2
complex, most of the experiments described herein followed the
localization of
1.
View larger version (60K):
[in a new window]
Fig. 1.
Co-localization of s,
1, and
2. Expression vectors
encoding
1 and
2 were transfected into
HEK293 cells in the absence (a and b) or presence
(c-h) of pcDNA3 containing
s. Cells were
fixed, and expressed proteins were visualized by immunofluorescent
staining as described under "Experimental Procedures." The
antibodies utilized are as follows: for
s, anti-HA
polyclonal and Alexa 488 anti-rabbit antibodies (c) or
anti-HA monoclonal and Alexa 488 anti-mouse antibodies (e);
for
1, anti-Myc monoclonal and Alexa 594 anti-mouse
antibodies (a, d, and g); and for
2, anti-
2 polyclonal and Alexa 488 (b and h) or 594 (f) anti-rabbit
antibodies.
Replacement of Cysteine with Serine in the CAAX Motif of the
2 Subunit Resulted in Loss of PM Targeting but Not
Binding--
The C terminus of the
2 subunit contains
the CAAX motif, specifically the sequence of cysteine,
alanine, isoleucine, and leucine. The cysteine is modified with a
20-carbon geranylgeranyl isoprenoid. We substituted a serine for the
cysteine and transiently expressed
2C68S in HEK293 cells
in conjunction with wild type
1 in the presence and
absence of wild type
s. In immunofluorescent staining,
little
1
2C68S was found at the PM
regardless of
s expression (Fig.
2A). To test whether poor PM
localization of
1
2C68S, when expressed
with
s, resulted from an inability to form a
heterotrimer, the
1
2 dimer was pulled
down with Ni-NTA beads, taking advantage of an N-terminal hexahistidine
tag on
1, and immunoblotted for the
s
subunit.
1
2 was able to efficiently pull
down
s (Fig. 2B, lane 3).
Similarly, the
1
2C68S dimer pulled down
the
s subunit (Fig. 2B, lane
6), implying that
s and
1
2C68S are capable of assembling a
heterotrimer. In this assay, efficient heterotrimer formation required
co-expression of all three components. When lysates from cells
expressing
1
2 or
1
2C68S were mixed with a lysate from
cells expressing only
s, heterotrimer formation was not
detected (Fig. 2B, lanes 4 and 7). The
results with
2C68S indicate that non-prenylated
2 can form a complex with wild type
s and
1, but the
1
2C68S dimer
was not localized at the PM.
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The 1
2 Complex Was Prenylated in the
Absence of the
s Subunit--
To determine whether
1
2 displayed very poor PM targeting
without the co-expressed
s because of inefficient lipid
modification of the
2 subunit, a prenylation assay was
carried out. Because HEK293 cells, in our hands, detached from the
bottom of culture plates upon treatment with mevastatin, a
hydroxymethylglutaryl-CoA reductase inhibitor, the experiments were
carried out using COS7 cells. Similar to its subcellular localization
in HEK293 cells,
1
2 is poorly targeted to
the PM in COS7 cells unless an
subunit is also co-expressed (13).
The
1 and
2 subunits were transiently transfected into COS7 cells in the absence or presence of the
s subunit. The transfected cells were labeled with
[3H]mevalolactone in the presence of mevastatin for
18 h. The
1
2 complexes were isolated
using Ni-NTA beads. Because the
1 subunit contains the
N-terminal hexahistidine tag, only
2 subunits that are
bound to
1 are pulled down in these experiments. The
level of isoprenoid incorporated into
2 was visualized
by fluorography and that of expression of
2 was assessed
by Western blotting. Mock/pcDNA3 transfection showed no nonspecific
uptake of the radioactivity (Fig. 2C, lane 1),
and as expected,
2C68S failed to incorporate radioactivity (Fig. 2C, lane 4). The
1
2 efficiently incorporated radioactive
isoprenoid in the presence of the
s subunit, and virtually no difference was seen in incorporation of radioactivity into
the
1
2 without the
s
subunit (Fig. 2C, lanes 2 and 3). Therefore, the defect in the PM localization of
1
2 when expressed in the absence of
was not due to failure of the
2 subunit to be
prenylated. In other words, the
1
2 dimer
is capable of being modified with the isoprenoid group in the absence
of the
subunit. It is therefore conceivable that lipid modification
takes place prior to trimer formation.
Prenylated 1
2 Dimer Was
Membrane-bound--
Without co-expressed
s, the
1
2 complex is prenylated but localized to
the PM very poorly. Next we examined the subcellular localization of
the prenylated
1
2 by a cell fractionation
assay. After transient transfection, cells were lysed in hypotonic
buffer, and the soluble and particulate fractions, representing
cytoplasmic and membrane fractions, were separated by
ultracentrifugation. Proteins in each fraction were analyzed by Western
blotting using anti-Myc monoclonal antibody to detect the
1 subunit of the
1
2 dimer.
With co-expressed
s, virtually all
1
2 complexes were found in the
particulate fraction (Fig.
3A, lane 2),
presumably tethered to the PM (Fig. 1d). When expressed
alone, the
1
2 dimers, which localized
very poorly at the PM (Fig. 1, a and b), were also found mostly in the particulate fraction (Fig.
3A, lane 4). This suggests that the prenylated
1
2 was targeted to membranes other than
the PM. To examine the intracellular localization of
1
2 more closely, we compared the
subcellular localization of
1
2 with an ER
marker protein using confocal microscopy.
1
2, when expressed alone, exhibited a
subcellular distribution virtually identical to the ER marker (Fig.
3B, a and b), whereas
1
2, when expressed with
s,
displayed PM localization that was clearly distinct from the ER (Fig.
3B, c and d). These results are thus consistent with a model in which the
1
2
dimer is geranylgeranylated in the cytosol by a cytoplasmic
geranylgeranyltransferase (14) and then targeted to ER, prior to
-dependent transit to the PM.
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Introduction of a Palmitoylation Site into 2 Allows
s-independent PM Targeting of
1
2--
It has been shown that
isoprenylation is necessary but not sufficient for PM targeting of the
Ras family of small GTPase, and a so-called second membrane targeting
signal is required for PM targeting (6, 7). H-Ras and N-Ras have been
shown to be palmitoylated at cysteines upstream of their
CAAX boxes. K-Ras possesses polybasic lysines flanking the
prenylcysteine. Of interest, Ste18p, the yeast
subunit, also is
palmitoylated at a cysteine next to the prenylcysteine, and
palmitoylation is necessary for avid PM membrane binding (8, 15). We
tested the possibility that the
1
2 dimer
becomes able to localize to the PM if
2 is bestowed with
a "second" membrane targeting signal by constructing
mutants
with a potential palmitoylation site. A phenylalanine residue at the 66 or 67 position of the
2 subunit was replaced with a
cysteine, based on the site of palmitoylation in H-Ras or Ste18p,
respectively (Fig. 4A). The
mutant
2 and wild type
1 were transiently
expressed in HEK293 cells. Unlike the
1
2 complex containing wild type
2 which displays
predominant intracellular staining and weak or no PM staining (Fig.
4B, a), the dimer with
2F66C or
2F67C showed strong PM localization without
overexpressed
s (Fig. 4B, b and
c). This result indicates that with a second signal the
1
2 dimer is capable of trafficking to the
PM in the absence of the
subunit. These results suggest that
requires a second membrane targeting signal for efficient PM
localization. Palmitate linked to
s may serve as the
second signal for the
1
2 dimer.
Consistent with this model,
1
2 fails to
localize efficiently at the PM when co-expressed with a
palmitoylation-deficient mutant of
s,
sC3S (Fig. 3B, f), or
q,
qC9S,C10S (not shown).
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1
2 Followed an
s
Artificially Directed to Mitochondria--
To examine further a
role for the
subunit in
localization, we utilized a strategy
described previously (11) to study protein-protein interactions: target
one protein to a location where it does not normally exist and assess
the ability of the artificially directed protein to be accompanied by
its partner protein. We targeted the
s subunit to
mitochondria and tested whether the
dimer follows. A
mitochondria targeting signal sequence derived from Mas70p/Tom70 was
fused to the N terminus of HA-tagged
s to generate
mito-
s. This peptide sequence has been shown to bring a
fused protein to the mitochondrial outer membrane without subsequent
import of the protein into the mitochondrial matrix (16). The
integrated protein is therefore anchored at the cytosolic face of the
mitochondrial outer membrane and is available to interact with other
proteins. A mitochondria localization vector, pEYFP-Mito, was used as a
mitochondrial marker. Wild type
s exhibited pronounced
PM staining, displaying little or no overlap with the mitochondrial
marker (Fig. 5, a-c). On the
other hand, mito-
s was co-localized with the
mitochondrial marker (Fig. 5, d-f). Overlaying of the two
pictures showed conclusively that mito-
s localized at
mitochondria, as demonstrated by the yellow color in Fig.
5f. When
1 and
2 were
expressed with mito-
s, the
1
2 dimer accompanied
mito-
s to mitochondria (Fig. 5, j and
k), as a superimposed image clearly demonstrates
co-localization (Fig. 5, l). In contrast, the
1
2 complex was found at the PM when
expressed with wild type
s (Fig. 5,
g-i).
|
Impaired Interaction of with
Prevented PM Localization
of
--
To address more directly the requirement for assembly
of the
heterotrimer in
localization, we tested the
subcellular localization of
-binding-deficient
mutants. Two
surfaces of
, the
switch interface and the
N-terminal
interface, contain important residues for interaction with the
subunit (17, 18). Mutation of the putative
-contacting residues in
the
subunit resulted in a decreased affinity for the
subunit
(12, 19). Based on previous findings, we introduced the mutations,
I80A, N88A/K89A, L117A, D228R, or D246S (note that Ile-80,
Asn-88, and Lys-89 are in the
N-terminal interface, and Leu-117,
Asp-228, and Asp-246 are in the
switch interface), into
Myc-His-tagged
1 and transiently expressed them in
conjunction with wild type
2 in HEK293 cells. All mutant
1
2 showed meager PM localization, similar
to wild type
1
2 (Fig.
6A, a). Just as
wild type
1
2 displayed much greater PM
localization when co-expressed with wild type
s, the
mutant
1
2 complex exhibited stronger PM
membrane targeting when expressed with
s (Fig.
6A, b). The ability of
s to
promote PM localization of the
-binding-deficient mutants of
1 suggests that the
1 mutants are not
completely unable to interact with
. Consistent with this
interpretation, others (12, 19) using these
1 mutants
observed varying degrees of loss of
binding, depending upon the
assay used.
|
Next, we expressed the 1 mutants and wild type
2 in conjunction with an
s mutant,
s IEK+, that contains mutations to five N-terminal amino
acids at the
binding interface. Our previous work (10)
demonstrated that this mutant lost its ability to localize to the PM
when expressed alone, but co-expression of wild type
restored
the PM localization of the
sIEK+ mutant. It was expected
that a combination of the
-binding-deficient mutant of
s and an
-binding-deficient mutant of
1 would result in more impaired heterotrimer formation.
Consistent with this prediction, a
1
2
complex containing
1D246S (Fig. 6A,
c) and, to lesser extent, ones with
1D228R and
1N88A/K89A (not shown) were poorly localized at the PM
when co-expressed with
sIEK+.
With these results, we sought to construct mutants with more severe
mutations, to generate ones that are less capable of binding to wild
type
. Three mutants were created by combining mutations in both
interfaces.
1NKD contains the mutations N88A, K89A, and
D246S;
1NKDD is like
1NKD with an
additional D228R mutation, and
1INKDD is further mutated
at I80A. To allay concerns that multiple mutations impede proper
folding of the
1 protein, we checked their ability to
bind the
2 subunit. The
1NKD and
1NKDD mutants showed
2 binding similar to
wild type
1 as assessed by Ni-NTA pull-down assay (Fig.
6B, lanes 1-3). Interestingly,
1INKDD, containing one additional mutation, failed to
associate with the
2 subunit (Fig. 6B,
lane 4), and thus
1INKDD was not analyzed further.
The abated capability of the mutants to interact with
s
was also confirmed by a Ni-NTA pull-down assay (Fig.
6B, lanes 6 and 7). Collectively, the
1NKD and
1NKDD mutants are correctly
folded yet substantially defective in association with
s. When these two mutants were expressed with wild type
2, the
1
2 complex was
localized to the PM poorly, similar to wild type
1 (Fig.
6C, a and c). Importantly,
co-expression of wild type
s did not promote PM
localization of the dimer containing either mutant (Fig.
6C, b and d). Expression of the
s subunit was confirmed by double staining of the
subunit in the same cells. Collectively, impaired interaction of the
dimer with the
subunit resulted in poor PM targeting of the
dimer. These results underscore the significance of proper heterotrimer
formation and indicate that the
subunit plays an important role in
PM targeting of the
dimer.
![]() |
DISCUSSION |
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Data presented here refine the requirements for PM targeting of
the G protein complex. In addition to demonstrating that isoprenylation of
is required but not sufficient, our results reveal a crucial role for the G protein
subunit. Thus, both heterotrimer assembly and lipid modifications function together to
promote proper PM localization of
.
Substitution of cysteine 68 with serine in the C terminus of
2 prevented attachment of isoprenoid to it, and
1
2C68S exhibited virtually no PM
localization, consistent with earlier immunofluorescence observations
in COS cells (20). The additional co-expression of
s
failed to promote PM localization of
1
2C68S (Fig. 2A), although
s strongly promoted PM localization of
1
2 (Fig. 1d) (9). In addition,
we demonstrated that the
2C68S mutant can form a
heterotrimer with co-expressed
s and
1
subunits as assessed by Ni-NTA pull-down assay (Fig. 2B).
Although prenylation of the
complex has been reported to
increase its affinity for the
subunit (14, 21), prenylation is not
a strict requirement for heterotrimer formation. Consistent with this,
none of the subunits in a crystallized
complex contained
lipid modifications; the C68S
mutant produced in Sf9 cells
was able to assemble with
and
subunits (17). The inability of
the
1
2C68S complex to localize to the PM
when co-expressed with
s, even though it is capable of
binding
s, implies that heterotrimer formation alone is
not sufficient for PM targeting of the
dimer.
Heterotrimer formation, however, appears to be necessary for
localization of , and several lines of evidence are consistent with a role for
in PM targeting of
. First, we demonstrated previously that transiently expressed
was poorly targeted to the
PM (9) and was found predominantly at intracellular membranes (Fig. 1,
a and b, Fig. 3B, b, and
Fig. 4B, a). However, co-expression of an
subunit promoted strong PM localization of the
dimer (9)
(Fig. 1, d and g, and Fig. 3B,
d). A recent report (13) confirmed these results using a green
fluorescent protein-tagged
in COS cells. Second, when
s was targeted to mitochondria the
1
2 subunits followed (Fig. 5). The
ability of
s, in this case misdirected to mitochondria,
to mistarget
is consistent with a prominent role for
subunits in guiding
to its appropriate cellular destination.
Third, we directly tested the effects of impaired assembly in
the subcellular localization of the
dimer by mutating putative
-binding residues in the
subunit. Currently available crystal
structure models of the
complex indicate that the
subunit
contacts the
subunit at two surfaces, termed the
N-terminal
interface and the
switch interface (17, 18). Others reported (12,
19) that introduction of a mutation into the
subunit in either
interface resulted in reduced ability to form a heterotrimer properly.
However, when we examined subcellular localization of mutant
1
2 complexes in which the
1 contained single mutations, or the double N88A/K89A
mutation,
s was able to promote efficient PM
localization of the mutant
. This is consistent with
demonstrations that such
1 mutants still retain some
ability to interact with
(12, 19). Nonetheless, a defect in PM
localization of mutant
1
2 was revealed
when an
-binding defective
1 mutant was expressed
with
2 and a previously described
-binding
defective
s mutant
sIEK+ (9) (Fig.
6A, c). Combination of the
-binding
defective
and the
-binding defective
impeded proper
heterotrimer assembly, resulting in poor PM targeting. We further
constructed the mutants
1NKD and
1NKDD
with combined mutations in both
-binding interfaces to achieve more
impaired interaction with wild type
subunit. Importantly,
co-expression of
s failed to promote PM localization of
1NKD or
1NKDD (Fig. 6C,
b and d). The
1 mutants were capable of
binding
2 (Fig. 6B), and thus the failure of
PM targeting was not due to misfolding of the mutant
1
subunit. Collectively, the results with
-binding defective
1 mutants clearly demonstrate that interaction of the
subunit with the
subunit is critical in PM targeting of the
dimer. To our knowledge, these results are the first to show explicit evidence of the significance of heterotrimer assembly in
localization at the PM.
Fourth, studies in model organisms indicate a role for in targeting
. In the yeast S. cerevisiae,
is defective in
localizing at the PM in an
subunit (Gpa1) null mutant (22).
Moreover, expression of the yeast
Gpa1p rescues PM localization of
a cytoplasmic
mutant in which the palmitoylation site in
is
mutated (15). In addition, a recent study of G protein localization in
Caenorhabditis elegans showed that depletion of an
subunit resulted in failure of the
subunits to localize properly
(23). In C. elegans embryos, GPB-1, the
subunit, and
GOA-1, the
i/o subunit, were found at the cell PM and on
microtubule asters. When expression of GOA-1 and GPA-16, the
widely expressed
subunit with redundant functions to GOA-1 in
C. elegans, was abrogated by RNA interference, GPB-1 lost
its aster and PM localization (23). The orientation role of the
subunit in
localization may be widespread.
Recent findings (24, 25) revealed that Ras undergoes prenylation and
then transits via intracellular membranes to the PM rather than moving
directly from the cytosol to the PM as was once thought. G protein
subunits may take a similar pathway to the PM. The enzymes that
catalyze proteolytic cleavage of the last three amino acids and
methylation of the carboxyl group of the prenylcysteine have been
cloned recently and identified as membrane-bound proteins at the ER
(26, 27). Thus, Ras is prenylated in the cytosol and then targeted to
the cytoplasmic face of the ER where additional C-terminal processing
takes place. The
dimer is similarly prenylated in the cytoplasm
and then presumably targeted to the ER to undergo subsequent
CAAX processing. Interaction with an
subunit does not
appear to be required for the initial step in
trafficking.
is localized to intracellular membranes in the absence of
co-expression (Fig. 3), and
undergoes prenylation equally well
in the absence or presence of
expression (Fig. 2C). It
has been known that, in addition to the CAAX processing, a
second signal in the hypervariable region is required for PM targeting
of Ras (6, 7); H-Ras and N-Ras become palmitoylated at cysteines
upstream of the prenylcysteine, whereas K-Ras contains a polylysine
sequence adjacent to the prenylcysteine. Unlike Ras, none of the human
subunits contain potential palmitoylation residues or a stretch of
basic residues adjacent to the prenylcysteine. However, we demonstrate
that introduction of a potential palmitoylation site into
2 resulted in PM localization of
1
2 and obviated the requirement for
co-expression of
s (Fig. 4B, b and
c). This indicates that if the
subunit is conferred a
second signal, the dimer becomes able to transit to the PM alone.
Furthermore, it is conceivable that the
subunit may function as a
"provider" of the second signal, rendering its palmitate to the
complex. Consistent with this proposal,
1
2 fails to localize efficiently at the
PM when co-expressed with palmitoylation-deficient mutants of
(Fig.
3B, f) (13).
Not only does require
, as described here, but several
reports (28, 29) have demonstrated that
depends on binding to
for its PM localization. Expression of
recovers
palmitoylation and PM localization of non-myristoylated G2A mutants of
i and
z. Furthermore, inhibiting
and
interaction by expression of a
-sequestering protein (30)
or by mutating
contact sites in
subunits decreases
palmitoylation and PM localization of
z (11) or
s (10). Targeting of
1
2 to
mitochondria through a mito-
2 mutant also directs
co-expressed
z to mitochondria (11). This last result,
taken together with our reciprocal results showing that
mito-
s can direct
1
2 to
mitochondria, is consistent with the idea that both
and
rely
on heterotrimer formation to reach their destination.
Demonstrations of a role for in the PM targeting of
are not
incompatible with the results presented in this report. The reciprocal
requirement for heterotrimer assembly is most consistent with a model
in which heterotrimer formation occurs intracellularly prior to transit
of
to the PM. Thus, if heterotrimer formation must occur
before
or
can proceed to the PM, both
and
would
exhibit a requirement for binding to their partner in order to achieve
PM localization. On the other hand, the reciprocal requirement for
and
interaction is more difficult to understand in the context
of a model in which
and
traffic separately to the PM and
wait there for their respective partners, since such a model implies
that at least one of the
or
traffics to the PM and is stably
anchored there independent of binding to its partner. Although our
results are more consistent with the proposal that heterotrimer
formation occurs prior to PM localization, we cannot rule out that
and
traffic separately to the PM but are each rapidly recycled
to intracellular membranes unless heterotrimer formation occurs at the
PM.
Where do and
initially interact? Recently, heterotrimer
assembly at Golgi was suggested based on co-localization at the Golgi
of
and a palmitoylation-defective
i2 (13).
However, in the rat exocrine pancreas the
subunit was not found on
Golgi membranes, whereas various
subunits were detected there (31). Meanwhile, a palmitoyltransferase for Ras was found in ER membranes (32, 33). The involvement of the Golgi in G protein trafficking and the
location of a relevant palmitoyltransferase remain to be further investigated.
An emerging model of heterotrimer trafficking to the PM proposes the
following. Newly synthesized and
rapidly form a dimer, and the
C terminus of the
subunit is modified with an isoprenyl group in
the cytosol. Subsequently, the dimer transits to the ER where its
prenylated CAAX sequence is further processed. Finally, at
intracellular membranes, the
and
subunits form a
heterotrimer complex, and then
traffic to the PM together,
utilizing at least palmitate and isoprenoid as two-platoon membrane
targeting signals. Forming the heterotrimer prior to reaching the PM
may confer "heterotrimer-specific" localization and may be crucial to maintain the proper stoichiometry of
to
.
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ACKNOWLEDGEMENTS |
---|
We thank Janice Buss and John Stickney for valuable advice on the prenylation assay, and Maurine Linder, Daniel Evanko, and Manimekalai Thiyagarajan for critical reading of the manuscript and helpful comments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM56444 (to P. B. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology
and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., 839 BLSB, Philadelphia, PA 19107. Tel.: 215-503-3137; Fax:
215-923-2117; E-mail: P_Wedegaertner@mail.jci.tju. edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M213239200
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ABBREVIATIONS |
---|
The abbreviations used are: G protein, guanine nucleotide-binding protein; PM, plasma membrane; ER, endoplasmic reticulum; HEK293 cells, human embryonic kidney cells; COS7, African green monkey kidney cells; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline.
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