Heterotrimer Formation, Together with Isoprenylation, Is Required for Plasma Membrane Targeting of Gbeta gamma *

Satoshi Takida and Philip B. WedegaertnerDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nascent beta  and gamma  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 beta 1gamma 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 alpha  subunit allows strong PM localization of the beta 1gamma 2. Furthermore, C-terminal isoprenylation of the gamma  subunit is necessary but not sufficient for PM localization of beta 1gamma 2. Isoprenylation of gamma 2 and localization of beta 1gamma 2 to the ER occurs independently of alpha  expression. Efficient PM localization of beta 1gamma 2 in the absence of co-expressed alpha  is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into gamma 2. When a mutant of alpha s is targeted to mitochondria, beta 1gamma 2 follows, consistent with an important role for alpha  in promoting subcellular localization of beta gamma . Furthermore, we directly demonstrate the requirement for alpha  by showing that disruption of heterotrimer formation by the introduction of alpha  binding mutations into beta 1 impedes PM targeting of beta 1gamma 2. The results indicate that two membrane targeting signals, lipid modification and alpha  binding, make concerted contributions to PM localization of beta gamma .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins1 are composed of alpha  and beta gamma subunits. The beta gamma complex only dissociates when denatured and hence is a functional monomer under physiological conditions. Upon receptor activation the beta gamma dimer is freed from GTP-bound alpha  and relays signals to downstream molecules until it reassociates with GDP-bound alpha , 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 alpha  subunit and isoprenylation for the gamma  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.

Mechanisms underlying the PM targeting of the alpha  subunit have been studied in some detail (1-3). The available data suggest a model in which myristoylation and/or binding to beta gamma subunits constitutes an initial membrane targeting signal for the alpha  subunit. Subsequently, palmitoylation functions as a second signal that specifies localization to the PM. In contrast to the alpha  subunit, relatively less is known about how beta gamma 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 gamma  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 gamma  subunits thus far identified, gamma 1, gamma 9, and gamma 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 gamma  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 gamma  subunit in Saccharomyces cerevisiae, is modified with palmitate at the cysteine immediately next to the prenylcysteine (8). However, none of the human gamma  have such cysteines or a stretch of basic residues flanking its CAAX motif.

Previously we found that the beta gamma complex was localized poorly to the PM when transiently expressed in HEK293 cells, whereas co-expression of the alpha  subunit led to strong PM localization of beta gamma (9). This suggests that the complete information required for efficient PM targeting of beta gamma is not contained within the beta gamma dimer, and interaction of the beta gamma dimer with the alpha  subunit is critical for PM targeting of beta gamma . In this report, we examined the importance of the alpha  subunit in PM targeting of beta gamma . We show that isoprenylation of the gamma  subunit is necessary but not sufficient for PM localization of beta gamma , and expression of alpha  is not required for gamma  isoprenylation. We demonstrate that introduction of an additional membrane targeting signal into the gamma  subunit can overcome the reliance of beta gamma on alpha  for PM targeting. In addition, beta gamma accompanies an alpha  subunit mis-targeted to mitochondria. Finally, we present the first direct test of the necessity for heterotrimer assembly for PM localization of beta gamma by demonstrating that alpha  binding-deficient mutants of beta gamma fail to localize to the PM, even when co-expressed with alpha . The results presented herein are consistent with a model in which both heterotrimer assembly and lipid modifications, working in concert, target beta gamma and alpha  to the PM.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 alpha s (HA-tagged) and beta -binding-deficient mutant alpha sIEK+(HA-tagged) were described previously (10). The expression vector for beta 1 (Myc- and His-tagged) was provided by David P. Siderovski (University of North Carolina) (9). alpha -Binding defective beta 1 mutants, beta 1I80A, beta 1N88A/K89A, beta 1L117A, beta 1D228R, beta 1D246S, beta 1N88A/K89A/D246S (hereafter named beta 1NKD), beta 1N88A/K89A/D228R/D246S (named beta 1NKDD), and beta 1I80A/N88A/K89A/D228R/D246S (named beta 1INKDD), were created using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and Myc-His tagged beta 1 in pcDNA3.1 as the template. Non-tagged gamma 2 was described previously (9). gamma 2 (Myc-tagged) and gamma 2MITO in pcDNA3 (11) were provided by Henry R. Bourne (University of California, San Francisco). gamma 2F66C, gamma 2F67C, and gamma 2C68S were made from gamma 2 (no tag) using the QuickChange kit and so was Myc-tagged gamma 2C68S. A mitochondrial targeting sequence was excised from gamma 2MITO and inserted into wild type alpha s (HA-tagged) to create alpha s fused with mitochondrial targeting sequence (mito-alpha s). pcDNA3 containing His-tagged beta 1 was provided by Tohru Kozasa (University of Illinois, Chicago).

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 alpha :beta :gamma 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.

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 beta 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 beta 1.

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 beta 1 and non-tagged gamma 2 without or with HA-tagged alpha 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 beta gamma 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 gamma 2 subunit was detected by immunoblotting. Note that because an anti-gamma 2 polyclonal antibody did not recognize gamma 2C68S, His-tagged beta 1 (12) and Myc-tagged gamma 2C68S were used for the alpha sbeta 1gamma 2C68S control sample, and gamma 2C68S was detected on an immunoblot with an anti-Myc monoclonal antibody.

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-gamma 2 rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we focused on the alpha sbeta 1gamma 2 G protein heterotrimer. Immunofluorescence microscopy was utilized to examine the subcellular localization of the alpha  and beta gamma subunits. It has been shown that alpha s is predominantly localized to the PM when expressed alone (9, 10). As described by us previously (10), when beta 1 and gamma 2 were expressed together in HEK293 cells very little PM localization was observed (Fig. 1, a and b). However, when alpha s was co-expressed with beta 1 and gamma 2, alpha s and beta 1 strongly co-localized at the PM (Fig. 1, c and d). Expression of alpha q also strongly promoted PM localization of beta 1gamma 2 (not shown). gamma 2 displayed PM localization when alpha s, beta 1, and gamma 2 were all expressed together, but gamma 2 was also found intracellularly and not co-localizing with alpha s or beta 1 (Fig. 1, e-h). Apparently some of the gamma 2 did not form a dimer with beta 1. Because localization of beta 1 rather than gamma 2 appeared to be a better representative of the beta 1gamma 2 complex, most of the experiments described herein followed the localization of beta 1.


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Fig. 1.   Co-localization of alpha s, beta 1, and gamma 2. Expression vectors encoding beta 1 and gamma 2 were transfected into HEK293 cells in the absence (a and b) or presence (c-h) of pcDNA3 containing alpha s. Cells were fixed, and expressed proteins were visualized by immunofluorescent staining as described under "Experimental Procedures." The antibodies utilized are as follows: for alpha s, anti-HA polyclonal and Alexa 488 anti-rabbit antibodies (c) or anti-HA monoclonal and Alexa 488 anti-mouse antibodies (e); for beta 1, anti-Myc monoclonal and Alexa 594 anti-mouse antibodies (a, d, and g); and for gamma 2, anti-gamma 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 gamma 2 Subunit Resulted in Loss of PM Targeting but Not alpha  Binding-- The C terminus of the gamma 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 gamma 2C68S in HEK293 cells in conjunction with wild type beta 1 in the presence and absence of wild type alpha s. In immunofluorescent staining, little beta 1gamma 2C68S was found at the PM regardless of alpha s expression (Fig. 2A). To test whether poor PM localization of beta 1gamma 2C68S, when expressed with alpha s, resulted from an inability to form a heterotrimer, the beta 1gamma 2 dimer was pulled down with Ni-NTA beads, taking advantage of an N-terminal hexahistidine tag on beta 1, and immunoblotted for the alpha s subunit. beta 1gamma 2 was able to efficiently pull down alpha s (Fig. 2B, lane 3). Similarly, the beta 1gamma 2C68S dimer pulled down the alpha s subunit (Fig. 2B, lane 6), implying that alpha s and beta 1gamma 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 beta 1gamma 2 or beta 1gamma 2C68S were mixed with a lysate from cells expressing only alpha s, heterotrimer formation was not detected (Fig. 2B, lanes 4 and 7). The results with gamma 2C68S indicate that non-prenylated gamma 2 can form a complex with wild type alpha s and beta 1, but the beta 1gamma 2C68S dimer was not localized at the PM.


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Fig. 2.   Isoprenylation of gamma 2 is required for alpha s-dependent PM targeting of beta 1gamma 2, but alpha s does not affect isoprenylation of gamma 2. A, HEK293 cells were transfected with a plasmid encoding gamma 2C68S in conjunction with expression vectors for alpha s and beta 1. 48 h after transfection, cells were fixed, and immunofluorescent staining for beta 1 was carried out using an anti-Myc monoclonal antibody and an Alexa 594 anti-mouse antibody. B, HEK293 cells transiently expressing alpha s (lane 1), beta 1 and gamma 2 (lane 2), alpha s, beta 1, and gamma 2 (lane 3), beta 1 and gamma 2C68S (lane 5), or alpha s, beta 1, and gamma 2C68S (lane 6) were lysed and subjected to a Ni-NTA pull-down assay, utilizing an N-terminal hexahistidine tag on beta 1, as described under "Experimental Procedures." In some cases, lysates were combined as indicated before the Ni-NTA pull-down assay (lane 4 and 7). Eluates were analyzed for the presence of alpha s by SDS-PAGE and Western blotting using an anti-HA monoclonal antibody. C, COS7 cells were transiently transfected with pcDNA3 alone (lane 1) or with expression vectors encoding beta 1 and gamma 2 (lane 2), alpha s, beta 1, and gamma 2 (lane 3), or alpha s, beta 1, and gamma 2C68S (lane 4). After labeling for 18 h with [3H]mevalolactone, cells were lysed, and beta 1gamma 2 was pulled down with Ni-NTA beads. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was exposed to a film at -80 °C (upper panel). Subsequently, the membrane was subjected to Western blotting for the gamma 2 subunit (lower panel).

The beta 1gamma 2 Complex Was Prenylated in the Absence of the alpha s Subunit-- To determine whether beta 1gamma 2 displayed very poor PM targeting without the co-expressed alpha s because of inefficient lipid modification of the gamma 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, beta 1gamma 2 is poorly targeted to the PM in COS7 cells unless an alpha  subunit is also co-expressed (13). The beta 1 and gamma 2 subunits were transiently transfected into COS7 cells in the absence or presence of the alpha s subunit. The transfected cells were labeled with [3H]mevalolactone in the presence of mevastatin for 18 h. The beta 1gamma 2 complexes were isolated using Ni-NTA beads. Because the beta 1 subunit contains the N-terminal hexahistidine tag, only gamma 2 subunits that are bound to beta 1 are pulled down in these experiments. The level of isoprenoid incorporated into gamma 2 was visualized by fluorography and that of expression of gamma 2 was assessed by Western blotting. Mock/pcDNA3 transfection showed no nonspecific uptake of the radioactivity (Fig. 2C, lane 1), and as expected, gamma 2C68S failed to incorporate radioactivity (Fig. 2C, lane 4). The beta 1gamma 2 efficiently incorporated radioactive isoprenoid in the presence of the alpha s subunit, and virtually no difference was seen in incorporation of radioactivity into the beta 1gamma 2 without the alpha s subunit (Fig. 2C, lanes 2 and 3). Therefore, the defect in the PM localization of beta 1gamma 2 when expressed in the absence of alpha  was not due to failure of the gamma 2 subunit to be prenylated. In other words, the beta 1gamma 2 dimer is capable of being modified with the isoprenoid group in the absence of the alpha  subunit. It is therefore conceivable that lipid modification takes place prior to trimer formation.

Prenylated beta 1gamma 2 Dimer Was Membrane-bound-- Without co-expressed alpha s, the beta 1gamma 2 complex is prenylated but localized to the PM very poorly. Next we examined the subcellular localization of the prenylated beta 1gamma 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 beta 1 subunit of the beta 1gamma 2 dimer. With co-expressed alpha s, virtually all beta 1gamma 2 complexes were found in the particulate fraction (Fig. 3A, lane 2), presumably tethered to the PM (Fig. 1d). When expressed alone, the beta 1gamma 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 beta 1gamma 2 was targeted to membranes other than the PM. To examine the intracellular localization of beta 1gamma 2 more closely, we compared the subcellular localization of beta 1gamma 2 with an ER marker protein using confocal microscopy. beta 1gamma 2, when expressed alone, exhibited a subcellular distribution virtually identical to the ER marker (Fig. 3B, a and b), whereas beta 1gamma 2, when expressed with alpha 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 beta 1gamma 2 dimer is geranylgeranylated in the cytosol by a cytoplasmic geranylgeranyltransferase (14) and then targeted to ER, prior to alpha -dependent transit to the PM.


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Fig. 3.   beta 1gamma 2 localizes to intracellular membranes in the absence of alpha s co-expression. A, expression plasmids containing beta 1 and gamma 2 were transiently transfected in HEK293 cells with (lanes 1 and 2) or without (lanes 3 and 4) pcDNA3 encoding alpha s. The subunits were separated into soluble (S) (lanes 1 and 3) and particulate (P) (lanes 2 and 4) fractions as described under "Experimental Procedures." beta 1 was detected in an immunoblot using an anti-Myc monoclonal antibody. B, beta 1 and gamma 2 were transiently expressed in HEK293 cells in the absence (a and b) or presence of alpha s (c and d) or alpha sC3S (e and f). pEYFP vector encoding a partial protein from infectious bronchitis virus was co-expressed as an ER marker (a, c, and e). Transfected beta 1gamma 2 was visualized by immunofluorescent staining using anti-Myc monoclonal antibody directed against Myc-His-tagged beta 1 and Alexa 594 anti-mouse antibody (b, d, and f). Images were recorded using confocal laser scanning microscopy, as described under "Experimental Procedures." Bar, 10 µm.

Introduction of a Palmitoylation Site into gamma 2 Allows alpha s-independent PM Targeting of beta 1gamma 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 gamma  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 beta 1gamma 2 dimer becomes able to localize to the PM if gamma 2 is bestowed with a "second" membrane targeting signal by constructing gamma  mutants with a potential palmitoylation site. A phenylalanine residue at the 66 or 67 position of the gamma 2 subunit was replaced with a cysteine, based on the site of palmitoylation in H-Ras or Ste18p, respectively (Fig. 4A). The mutant gamma 2 and wild type beta 1 were transiently expressed in HEK293 cells. Unlike the beta 1gamma 2 complex containing wild type gamma 2 which displays predominant intracellular staining and weak or no PM staining (Fig. 4B, a), the dimer with gamma 2F66C or gamma 2F67C showed strong PM localization without overexpressed alpha s (Fig. 4B, b and c). This result indicates that with a second signal the beta 1gamma 2 dimer is capable of trafficking to the PM in the absence of the alpha  subunit. These results suggest that beta gamma requires a second membrane targeting signal for efficient PM localization. Palmitate linked to alpha s may serve as the second signal for the beta 1gamma 2 dimer. Consistent with this model, beta 1gamma 2 fails to localize efficiently at the PM when co-expressed with a palmitoylation-deficient mutant of alpha s, alpha sC3S (Fig. 3B, f), or alpha q, alpha qC9S,C10S (not shown).


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Fig. 4.   Introduction of a second membrane targeting signal into gamma 2. A, the C termini of H-Ras, Step18p, and human gamma 2 subunit are aligned. Prenylcysteines are underlined and palmitoylcysteines in H-Ras and Ste18p are shown in boldface and underlined. The substituted cysteine for phenylalanine in the 66- or 67-position of gamma 2 is shown in boldface. B, gamma 2 (a), gamma 2F66C (b), or gamma 2F67C (c) were transiently expressed in HEK293 cells in conjunction with beta 1. Immunofluorescence staining for beta 1 was carried out using anti-Myc monoclonal and Alexa 594 anti-mouse antibodies.

beta 1gamma 2 Followed an alpha s Artificially Directed to Mitochondria-- To examine further a role for the alpha  subunit in beta gamma 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 alpha s subunit to mitochondria and tested whether the beta gamma dimer follows. A mitochondria targeting signal sequence derived from Mas70p/Tom70 was fused to the N terminus of HA-tagged alpha s to generate mito-alpha 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 alpha s exhibited pronounced PM staining, displaying little or no overlap with the mitochondrial marker (Fig. 5, a-c). On the other hand, mito-alpha s was co-localized with the mitochondrial marker (Fig. 5, d-f). Overlaying of the two pictures showed conclusively that mito-alpha s localized at mitochondria, as demonstrated by the yellow color in Fig. 5f. When beta 1 and gamma 2 were expressed with mito-alpha s, the beta 1gamma 2 dimer accompanied mito-alpha s to mitochondria (Fig. 5, j and k), as a superimposed image clearly demonstrates co-localization (Fig. 5, l). In contrast, the beta 1gamma 2 complex was found at the PM when expressed with wild type alpha s (Fig. 5, g-i).


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Fig. 5.   Mito-alpha s targets beta 1gamma 2 to mitochondria. HEK293 cells were transfected with expression vectors for beta 1 and gamma 2 in conjunction with the mitochondria marker pEYFP-Mito and either alpha s (a-c and g-i) or mito-alpha s (d-f and j-l). 48 h after transfection, immunofluorescent staining was performed. alpha s was visualized (b and e) using anti-HA polyclonal and Alexa 594 anti-rabbit antibodies. To visualize beta 1 of the beta 1gamma 2 dimer, an anti-Myc monoclonal antibody and Alexa 594 anti-mouse antibody were used for immunofluorescence (h and k). EYFP-Mito was visualized by fluorescence microscopy of the YFP (a, d, g, and j). Images were merged (c, f, i, and l) to show two-color staining.

Impaired Interaction of beta gamma with alpha  Prevented PM Localization of beta gamma -- To address more directly the requirement for assembly of the alpha beta gamma heterotrimer in beta gamma localization, we tested the subcellular localization of alpha -binding-deficient beta  mutants. Two surfaces of beta , the alpha  switch interface and the alpha  N-terminal interface, contain important residues for interaction with the alpha  subunit (17, 18). Mutation of the putative alpha -contacting residues in the beta  subunit resulted in a decreased affinity for the alpha  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 alpha  N-terminal interface, and Leu-117, Asp-228, and Asp-246 are in the alpha  switch interface), into Myc-His-tagged beta 1 and transiently expressed them in conjunction with wild type gamma 2 in HEK293 cells. All mutant beta 1gamma 2 showed meager PM localization, similar to wild type beta 1gamma 2 (Fig. 6A, a). Just as wild type beta 1gamma 2 displayed much greater PM localization when co-expressed with wild type alpha s, the mutant beta 1gamma 2 complex exhibited stronger PM membrane targeting when expressed with alpha s (Fig. 6A, b). The ability of alpha s to promote PM localization of the alpha -binding-deficient mutants of beta 1 suggests that the beta 1 mutants are not completely unable to interact with alpha . Consistent with this interpretation, others (12, 19) using these beta 1 mutants observed varying degrees of loss of alpha  binding, depending upon the assay used.


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Fig. 6.   alpha -Binding mutations in beta 1 impede PM targeting of beta 1gamma 2. A, beta 1D246S was transfected into HEK293 cells in conjunction with gamma 2 in the absence (a) or presence of either wild type alpha s (b) or alpha sIEK+ (c). Immunofluorescent staining was performed to detect beta 1 of the beta 1gamma 2 dimer using anti-Myc monoclonal and Alexa 594 anti-mouse antibodies. B, HEK293 cells transiently expressing beta 1 and gamma 2 (lane 1), beta 1NKD and gamma 2 (lane 2), beta 1NKDD and gamma 2 (lane 3), beta 1INKDD and gamma 2 (lane 4), alpha s, beta 1, and gamma 2 (lane 5), alpha s, beta 1NKD, and gamma 2 (lane 6), or alpha s, beta 1NKDD, and gamma 2 (lane 7) were lysed 48 h after transfection, and beta 1gamma 2 dimers were pulled down using Ni-NTA beads by virtue of the hexahistidine tag on Myc-His-tagged beta 1 or beta 1 mutants. Samples were separated by SDS-PAGE followed by immunoblotting using anti-gamma 2 antibody (lanes 1-4) or anti-HA antibody (lanes 5-7) (upper panel). The level of gamma 2 or alpha s expression in the lysates was assessed by Western blotting (lower panel). C, expression vectors encoding the following proteins were transfected into HEK293 cells: beta 1NKD and gamma 2 (a); alpha s, beta 1NKD, and gamma 2 (b); beta 1NKDD and gamma 2 (c); alpha s, beta 1NKDD, and gamma 2 (d). As above, localization of the beta 1gamma 2 dimer was visualized by immunofluorescent staining using anti-Myc monoclonal and Alexa 594 anti-mouse antibodies.

Next, we expressed the beta 1 mutants and wild type gamma 2 in conjunction with an alpha s mutant, alpha s IEK+, that contains mutations to five N-terminal amino acids at the beta gamma 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 beta gamma restored the PM localization of the alpha sIEK+ mutant. It was expected that a combination of the beta gamma -binding-deficient mutant of alpha s and an alpha -binding-deficient mutant of beta 1 would result in more impaired heterotrimer formation. Consistent with this prediction, a beta 1gamma 2 complex containing beta 1D246S (Fig. 6A, c) and, to lesser extent, ones with beta 1D228R and beta 1N88A/K89A (not shown) were poorly localized at the PM when co-expressed with alpha sIEK+.

With these results, we sought to construct beta  mutants with more severe mutations, to generate ones that are less capable of binding to wild type alpha . Three mutants were created by combining mutations in both interfaces. beta 1NKD contains the mutations N88A, K89A, and D246S; beta 1NKDD is like beta 1NKD with an additional D228R mutation, and beta 1INKDD is further mutated at I80A. To allay concerns that multiple mutations impede proper folding of the beta 1 protein, we checked their ability to bind the gamma 2 subunit. The beta 1NKD and beta 1NKDD mutants showed gamma 2 binding similar to wild type beta 1 as assessed by Ni-NTA pull-down assay (Fig. 6B, lanes 1-3). Interestingly, beta 1INKDD, containing one additional mutation, failed to associate with the gamma 2 subunit (Fig. 6B, lane 4), and thus beta 1INKDD was not analyzed further. The abated capability of the mutants to interact with alpha s was also confirmed by a Ni-NTA pull-down assay (Fig. 6B, lanes 6 and 7). Collectively, the beta 1NKD and beta 1NKDD mutants are correctly folded yet substantially defective in association with alpha s. When these two mutants were expressed with wild type gamma 2, the beta 1gamma 2 complex was localized to the PM poorly, similar to wild type beta 1 (Fig. 6C, a and c). Importantly, co-expression of wild type alpha s did not promote PM localization of the dimer containing either mutant (Fig. 6C, b and d). Expression of the alpha s subunit was confirmed by double staining of the subunit in the same cells. Collectively, impaired interaction of the beta gamma dimer with the alpha  subunit resulted in poor PM targeting of the dimer. These results underscore the significance of proper heterotrimer formation and indicate that the alpha  subunit plays an important role in PM targeting of the beta gamma dimer.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Data presented here refine the requirements for PM targeting of the G protein beta gamma complex. In addition to demonstrating that isoprenylation of gamma  is required but not sufficient, our results reveal a crucial role for the G protein alpha  subunit. Thus, both heterotrimer assembly and lipid modifications function together to promote proper PM localization of beta gamma .

Substitution of cysteine 68 with serine in the C terminus of gamma 2 prevented attachment of isoprenoid to it, and beta 1gamma 2C68S exhibited virtually no PM localization, consistent with earlier immunofluorescence observations in COS cells (20). The additional co-expression of alpha s failed to promote PM localization of beta 1gamma 2C68S (Fig. 2A), although alpha s strongly promoted PM localization of beta 1gamma 2 (Fig. 1d) (9). In addition, we demonstrated that the gamma 2C68S mutant can form a heterotrimer with co-expressed alpha s and beta 1 subunits as assessed by Ni-NTA pull-down assay (Fig. 2B). Although prenylation of the beta gamma complex has been reported to increase its affinity for the alpha  subunit (14, 21), prenylation is not a strict requirement for heterotrimer formation. Consistent with this, none of the subunits in a crystallized alpha beta gamma complex contained lipid modifications; the C68S gamma  mutant produced in Sf9 cells was able to assemble with alpha  and beta  subunits (17). The inability of the beta 1gamma 2C68S complex to localize to the PM when co-expressed with alpha s, even though it is capable of binding alpha s, implies that heterotrimer formation alone is not sufficient for PM targeting of the beta gamma dimer.

Heterotrimer formation, however, appears to be necessary for localization of beta gamma , and several lines of evidence are consistent with a role for alpha  in PM targeting of beta gamma . First, we demonstrated previously that transiently expressed beta gamma 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 alpha  subunit promoted strong PM localization of the beta gamma dimer (9) (Fig. 1, d and g, and Fig. 3B, d). A recent report (13) confirmed these results using a green fluorescent protein-tagged gamma  in COS cells. Second, when alpha s was targeted to mitochondria the beta 1gamma 2 subunits followed (Fig. 5). The ability of alpha s, in this case misdirected to mitochondria, to mistarget beta gamma is consistent with a prominent role for alpha  subunits in guiding beta gamma to its appropriate cellular destination.

Third, we directly tested the effects of impaired alpha beta gamma assembly in the subcellular localization of the beta gamma dimer by mutating putative alpha -binding residues in the beta  subunit. Currently available crystal structure models of the alpha beta gamma complex indicate that the beta  subunit contacts the alpha  subunit at two surfaces, termed the alpha  N-terminal interface and the alpha  switch interface (17, 18). Others reported (12, 19) that introduction of a mutation into the beta  subunit in either interface resulted in reduced ability to form a heterotrimer properly. However, when we examined subcellular localization of mutant beta 1gamma 2 complexes in which the beta 1 contained single mutations, or the double N88A/K89A mutation, alpha s was able to promote efficient PM localization of the mutant beta gamma . This is consistent with demonstrations that such beta 1 mutants still retain some ability to interact with alpha  (12, 19). Nonetheless, a defect in PM localization of mutant beta 1gamma 2 was revealed when an alpha -binding defective beta 1 mutant was expressed with gamma 2 and a previously described beta gamma -binding defective alpha s mutant alpha sIEK+ (9) (Fig. 6A, c). Combination of the beta gamma -binding defective alpha  and the alpha -binding defective beta  impeded proper heterotrimer assembly, resulting in poor PM targeting. We further constructed the mutants beta 1NKD and beta 1NKDD with combined mutations in both alpha -binding interfaces to achieve more impaired interaction with wild type alpha  subunit. Importantly, co-expression of alpha s failed to promote PM localization of beta 1NKD or beta 1NKDD (Fig. 6C, b and d). The beta 1 mutants were capable of binding gamma 2 (Fig. 6B), and thus the failure of PM targeting was not due to misfolding of the mutant beta 1 subunit. Collectively, the results with alpha -binding defective beta 1 mutants clearly demonstrate that interaction of the beta gamma subunit with the alpha  subunit is critical in PM targeting of the beta gamma dimer. To our knowledge, these results are the first to show explicit evidence of the significance of heterotrimer assembly in beta gamma localization at the PM.

Fourth, studies in model organisms indicate a role for alpha  in targeting beta gamma . In the yeast S. cerevisiae, beta gamma is defective in localizing at the PM in an alpha  subunit (Gpa1) null mutant (22). Moreover, expression of the yeast alpha  Gpa1p rescues PM localization of a cytoplasmic beta gamma mutant in which the palmitoylation site in gamma  is mutated (15). In addition, a recent study of G protein localization in Caenorhabditis elegans showed that depletion of an alpha  subunit resulted in failure of the beta gamma subunits to localize properly (23). In C. elegans embryos, GPB-1, the beta  subunit, and GOA-1, the alpha i/o subunit, were found at the cell PM and on microtubule asters. When expression of GOA-1 and GPA-16, the widely expressed alpha  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 alpha  subunit in beta gamma 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 beta gamma 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 beta gamma dimer is similarly prenylated in the cytoplasm and then presumably targeted to the ER to undergo subsequent CAAX processing. Interaction with an alpha  subunit does not appear to be required for the initial step in beta gamma trafficking. beta gamma is localized to intracellular membranes in the absence of alpha  co-expression (Fig. 3), and beta gamma undergoes prenylation equally well in the absence or presence of alpha  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 gamma  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 gamma 2 resulted in PM localization of beta 1gamma 2 and obviated the requirement for co-expression of alpha s (Fig. 4B, b and c). This indicates that if the gamma  subunit is conferred a second signal, the dimer becomes able to transit to the PM alone. Furthermore, it is conceivable that the alpha  subunit may function as a "provider" of the second signal, rendering its palmitate to the beta gamma complex. Consistent with this proposal, beta 1gamma 2 fails to localize efficiently at the PM when co-expressed with palmitoylation-deficient mutants of alpha  (Fig. 3B, f) (13).

Not only does beta gamma require alpha , as described here, but several reports (28, 29) have demonstrated that alpha  depends on binding to beta gamma for its PM localization. Expression of beta gamma recovers palmitoylation and PM localization of non-myristoylated G2A mutants of alpha i and alpha z. Furthermore, inhibiting alpha  and beta gamma interaction by expression of a beta gamma -sequestering protein (30) or by mutating beta gamma contact sites in alpha  subunits decreases palmitoylation and PM localization of alpha z (11) or alpha s (10). Targeting of beta 1gamma 2 to mitochondria through a mito-gamma 2 mutant also directs co-expressed alpha z to mitochondria (11). This last result, taken together with our reciprocal results showing that mito-alpha s can direct beta 1gamma 2 to mitochondria, is consistent with the idea that both alpha  and beta gamma rely on heterotrimer formation to reach their destination.

Demonstrations of a role for beta gamma in the PM targeting of alpha  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 alpha beta gamma to the PM. Thus, if heterotrimer formation must occur before alpha  or beta gamma can proceed to the PM, both alpha  and beta gamma would exhibit a requirement for binding to their partner in order to achieve PM localization. On the other hand, the reciprocal requirement for alpha  and beta gamma interaction is more difficult to understand in the context of a model in which alpha  and beta gamma traffic separately to the PM and wait there for their respective partners, since such a model implies that at least one of the alpha  or beta gamma 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 alpha  and beta gamma traffic separately to the PM but are each rapidly recycled to intracellular membranes unless heterotrimer formation occurs at the PM.

Where do alpha  and beta gamma initially interact? Recently, heterotrimer assembly at Golgi was suggested based on co-localization at the Golgi of beta gamma and a palmitoylation-defective alpha i2 (13). However, in the rat exocrine pancreas the beta  subunit was not found on Golgi membranes, whereas various alpha  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 beta  and gamma  rapidly form a dimer, and the C terminus of the gamma  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 beta gamma and alpha  subunits form a heterotrimer complex, and then alpha beta gamma 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 alpha  to beta gamma .

    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.

    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.

Dagger 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

    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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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