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Originally published In Press as doi:10.1074/jbc.M602409200 on May 15, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20221-20232, July 21, 2006
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Role of the Chaperonin CCT/TRiC Complex in G Protein beta{gamma}-Dimer Assembly*

Christopher A. Wells, Jane Dingus, and John D. Hildebrandt1

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, March 14, 2006 , and in revised form, May 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gbeta{gamma} dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, 35S-labeled G{gamma} subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, 35S-labeled Gbeta subunits traveled at a high apparent molecular mass (~700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with G{gamma}. When translated G{gamma} was added to translated Gbeta, a new band of low apparent molecular mass (~50 kDa) was observed, which was labeled by either 35S-labeled Gbeta or G{gamma}, indicating that it is a dimer. Formation of the Gbeta{gamma} dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg2+. This inhibition led to increased association of Gbeta with CCT/TRiC. Although G{gamma} did not bind CCT/TRiC, addition of G{gamma} to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbeta{gamma} dimer formation by an ATP-dependent reaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human genome codes for nearly 700 G protein-coupled receptors (1, 2), which signal through heterotrimeric G proteins and mediate the effects of a vast array of hormones, neurotransmitters, sensory signals, and drugs (3, 4). G protein-coupled receptors transmit signals to downstream effectors by catalytically activating target G proteins (5, 6). Theoretically, at least, the G protein targets of the G protein-coupled receptors are as diverse as the receptors through their combinatorial formation from 16 G{alpha}, 5Gbeta, and 12 G{gamma} gene products (7). Signaling specificity of heterotrimers is affected both by the biochemistry of their protein-protein interactions and by cellular targeting mechanisms that direct placement of receptors, G proteins, and second messenger targets (7, 8). An important but poorly understood process that affects all aspects of the signaling specificity of the heterotrimeric G proteins is the synthesis and assembly of G protein heterotrimers.

Gbeta{gamma} dimer formation is an early event in G protein assembly (9, 10). Biochemically, the Gbeta{gamma} complex is a very stable dimer (11) that reversibly interacts with the G{alpha} subunit, dependent on the associated guanine nucleotide (5). Given the apparent stability of the Gbeta{gamma} dimer, its assembly is thought of as an all-ornone event; however, recent work indicates that the efficiency of dimer assembly in vitro is related to isoform-specific interactions of Gbeta (12). Gbeta is a member of a large family of WD repeat proteins (13) that contain 4–16 repeats of a core of ~40 amino acid. Gbeta has seven repeats that form a closed, toroid-shaped, seven-bladed, beta-propeller structure (14, 15). Folding of Gbeta into this structure requires G{gamma} (16) and the participation of as yet unidentified components of in vitro translation mixtures (17, 18). One candidate for mediating Gbeta folding and Gbeta{gamma} dimer formation is the chaperonin CCT/TRiC complex (18–20).

65–80% of newly translated proteins fold spontaneously. In eukaryotes, at least three systems of molecular chaperones help fold the other 20–35% of cellular proteins: the heat shock proteins Hsp40 and Hsp70, the Hsp90 system, and the chaperonins (19, 21, 22). Chaperonin complexes are found in prokaryotes (Group I) and eukaryotes (Group II). Group I is the GroEL·GroES complex, in which GroEL is formed by two rings of seven identical 57-kDa monomers that nonspecifically recognize proteins with exposed hydrophobic surfaces. The eukaryotic Group II chaperonins are called CCT (chaperonin containing TCP-1) or TRiC (TCP-1 Ring complex); TCP-1 is tailless complex polypeptide-1 (22). Eukaryotic CCT/TRiC is considerably more complex than GroEL, with eight different subunits that share 30% sequence identity, and it recognizes specific but poorly defined sequences in target proteins (19, 23, 24). CCT/TRiC was originally characterized in the folding of actin and tubulin, but is now recognized to be involved in the folding of many different proteins (19, 21, 22), a subset of which includes some WD repeat proteins (19, 25), although it is not involved in the folding of all WD repeat proteins (26).

Isolated CCT/TRiC subunits were identified as interacting partners of the yeast Gbeta homolog Ste4 in a high throughput screen of yeast genome coding regions (27), but this did not extend to functional characterization of these interactions or to association with the CCT/TRiC complex. More recently, small interfering RNA to TCP-1{alpha} was shown to decrease Gbeta{gamma} levels in cells (20), indicating the possibility that some cellular component involved in Gbeta{gamma} dimer formation is dependent on the function of CCT/TRiC, and this was proposed as a site of regulation of Gbeta{gamma} dimer formation. Despite these observations, there is no direct evidence that CCT/TRiC does or even could participate either in folding of Gbeta or in Gbeta{gamma} dimer formation. There is not even any information about whether eukaryotic Gbeta or Gbeta{gamma} interacts with CCT/TRiC. Here, we have studied the mechanism of Gbeta{gamma} dimer formation in rabbit reticulocyte lysate and describe the role of CCT/TRiC both in Gbeta folding and in Gbeta{gamma} dimer formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vectors—All Gbeta and FLAG-G{gamma} constructs were as described previously (12). All FLAG-Gbeta constructs were obtained from the Guthrie cDNA Resource Center (available at www.cdna.org).

In Vitro Translation and Transcription—In vitro translation of subunits was carried out using the TNT® Quick-Coupled Transcription/Translation System (manual no. TM045, Promega Corp.) as described previously (12). Any additions to the translation mixture were added before DNA and lysate.

Gbeta{gamma} Dimerization Assay by Immunoprecipitation—Quantitation of synthesized protein and the dimerization reaction were done as described previously (12).

Immunoprecipitation of Synthesized Subunits—Immunoprecipitation of the FLAG-tagged subunits from translations and/or dimerizations was carried out using agarose-conjugated anti-FLAG antibody (catalog no. A-2220, Sigma) with 5–20 µl of beads/sample. Beads were washed twice with 50 mM Tris (pH 7.4), 100 mM NaCl (Tris-buffered saline (TBS)2), 1 mg/ml bovine serum albumin, and 0.1% C12E10 (polyoxyethylene 10-lauryl ether) (TBSBC). The beads were blocked against non-specific binding by incubation for 1 h at room temperature in 10 volumes of TBSBC with 5% reticulocyte lysate and then washed twice with 50 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% C12E10 (TBSC). For immunoprecipitation, the sample was diluted 10–20-fold with TBSC; 5–20 µl of beads were added; and the samples were incubated for 1 h at room temperature. The samples were centrifuged, and the beads were resuspended in TBSC and transferred to a new tube to reduce nonspecific binding of Gbeta. The samples were washed a second time. Electrophoresis sample buffer was added to the beads to elute the bound proteins.

Electrophoresis—Electrophoresis was carried out by the Laemmli procedure using Criterion Tris-HCl precast gels (Bio-Rad). Native (nondenaturing) gel electrophoresis was carried out following a procedure modified from that of Hansen et al. (28) also using Criterion gels, but SDS and beta-mercaptoethanol were omitted from both sample and electrophoresis buffers. The gels were run at 4 °C at 0.4 A with an initial voltage of 140 V for 20–30 min and then 180–200 V for the remainder of the run. For autoradiography, the gel was washed twice for 15 min with 50% methanol and 10% acetic acid and then fixed for 5 min with 7% acetic acid, 7% methanol, and 1% glycerol before drying. Dried gels were exposed to film overnight at –80 °C or to a phosphor storage screen for 3–5 days at room temperature. The screen was imaged and analyzed using a GE Healthcare Storm instrument and ImageQuant software. Molecular mass standards for nondenaturing gels were from Amersham Biosciences (high molecular mass calibration kit) and included thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa).

Immunoblotting—Immunoblotting of gels was performed by transferring the gels to nitrocellulose using either a Bio-Rad Trans-Blot or Trans-Blot SD semidry transfer apparatus (29). The antibodies used were the following: rabbit anti-Gbeta{gamma} antibody BC1 (30); mouse monoclonal anti-FLAG (Sigma); rat monoclonal anti-TCP-1{alpha} (Calbiochem); and goat anti-TCP-1{alpha}/beta/{gamma}/{delta}/{epsilon}/{theta}/{eta}/{zeta} (Santa Cruz Biotechnology, Inc.). After transfer, the nitrocellulose was blocked with 20 mM Tris (pH 7.5), 150 mM NaCl, and 0.5% Tween 20 (TBST) with 5% nonfat dry milk and 1% bovine serum albumin, and the primary and secondary antibodies were diluted in TBST with 0.5% nonfat dry milk and 0.1% bovine serum. The primary antibody was incubated for 1–3 h and washed extensively, and then the secondary antibody was incubated for 1 h. Blots were visualized using chemiluminescence (SuperSignal West Femto, Pierce). For simultaneous visualization of two antibodies, transfers were blocked with Odyssey blocking buffer (LI-COR Biosciences) and incubated with rabbit polyclonal anti-FLAG and goat anti-TCP-1{alpha} antibodies diluted in Odyssey blocking buffer (1:1 with TBST containing 0.2% Tween 20). IRDye 700DX-conjugated donkey anti-rabbit and IRDye 800-conjugated donkey anti-goat secondary antibodies (Rockland Immunochemicals, Inc.) were diluted in Odyssey blocking buffer (1:1 with TBST containing 0.01% SDS). Immunoblots were visualized using an Odyssey infrared imaging system (LI-COR Biosciences).

Proteomics Analysis of Samples Recovered after SDS-PAGE—FLAG-tagged Gbeta1 was expressed in vitro as described previously (16) and immunoprecipitated with anti-FLAG beads. Immunoprecipitated protein was eluted from the beads with SDS sample buffer without beta-mercaptoethanol and was run on 4–20% Criterion gels. Gels were stained with Coomassie Blue and destained with 10% acetic acid. Bands were excised with a scalpel and further processed in a PerkinElmer Life Sciences MultiPROBE II HT EX liquid-handling robot equipped with a Millipore Montage In-Gel DigestZP manifold. Gel slices were sequentially equilibrated with 25 mM ammonium bicarbonate and 5% acetonitrile, then with 25 mM ammonium bicarbonate and 50% acetonitrile, and finally with 100% acetonitrile. Protein was digested by reconstituting dehydrated gel slices with 15 µl of 10 µg/ml trypsin in 25 mM ammonium bicarbonate and incubating for 3 h at 37°C. Recovered peptides were analyzed by MALDI-TOF/TOF on an Applied Biosystems 4700 proteomics analyzer. Mass spectra were generated from 3000–5000 laser shots, and the 10 most intense protein peaks were sequenced by tandem mass spectrometry (MS/MS). The lower and upper limits of mass detection were 1000 and 3000 Da, respectively. Data were analyzed with an ABI GPS Explorer 3.5 running Mascot Version 1.9.05 for peak identification (31). The minimum signal-to-noise ratio was 10.0, and assignments were based upon a precursor tolerance of 1.0 Da, although most assigned peaks had a tolerance of <0.2 Da. Two data sets were analyzed: one of 4981 records containing all NCBI non-redundant rabbit (Oryctolagus cuniculus) proteins and the other of 163,861 records containing all NCBI non-redundant rodent proteins. Significant matches were identified based upon a Mascot protein score with >95% confidence interval for identification of the protein contained in the library. For the rabbit data base, this included all identified peaks with protein scores >44, whereas for the rodent data base, this included peaks with protein scores >63.


Figure 1
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  		FIGURE 1.
Dimerization of Gbeta1, Gbeta2, Gbeta3, Gbeta4, and Gbeta5 with FLAG-G{gamma}3. A, 35S-labeled Gbeta1, Gbeta2, Gbeta3, Gbeta4, and Gbeta5 and FLAG-G{gamma}3 proteins were expressed in the reticulocyte lysate. Equal aliquots of the total translation mixture were run on a 10–20% gradient gel. A PhosphorImager image of the dried gel is shown. The approximate positions of molecular mass standards (in kilodaltons) are shown on the left. B, Gbeta1, Gbeta2, Gbeta3, Gbeta4, and Gbeta5 were incubated with FLAG-G{gamma}3 at a 2:1 molar ratio, and FLAG-G{gamma}3 and any associated Gbeta subunits were immunoprecipitated (lanes 6–11). Controls included Gbeta subunits alone without FLAG-G{gamma}3 (lanes 1–5) and FLAG-G{gamma}3 alone (lane 11). Samples were analyzed as described for A, but on an 8–16% gradient gel. The approximate positions of molecular mass standards (in kilodaltons) are shown on the right.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gbeta{gamma} Dimer Formation between Gbeta Isoforms and FLAG-G{gamma}3—Gbeta1, Gbeta2, Gbeta3, Gbeta4, Gbeta5, and FLAG-G{gamma}3 were synthesized in the reticulocyte lysate in the presence of [35S]methionine (Fig. 1A), and quantitated as described (12). Gbeta and FLAG-G{gamma}3 were added together at a 2:1 stoichiometry, allowed to form dimers for 2 h at 37°C, and then immunoprecipitated with anti-FLAG beads. As shown in Fig. 1B, Gbeta1, Gbeta2, and Gbeta4 were easily co-immunoprecipitated with FLAG-G{gamma}3, indicating that Gbeta1{gamma}3, Gbeta2{gamma}3, and Gbeta4{gamma}3 dimers readily form. In contrast, no Gbeta5 was detectable in immunoprecipitates of FLAG-G{gamma}3 co-incubated with this Gbeta, indicating that Gbeta5 forms dimers very poorly with G{gamma}3. In immunoprecipitates with Gbeta3, much less Gbeta3 was present (relative to G{gamma}3) than was seen with Gbeta1, Gbeta2, or Gbeta4, but Gbeta3 was clearly present as compared with Gbeta5 or the control lanes. These results agree with our recent characterization of Gbeta{gamma} dimer association (12), which showed that Gbeta1 and Gbeta4 readily form dimers with all G{gamma} isoforms; that Gbeta2 selectively forms dimers at good efficiency with most G{gamma} isoforms, but not G{gamma}1, G{gamma}8c, G{gamma}11, or G{gamma}13; that Gbeta5 does not form dimers in the assay with any G{gamma} isoforms; and that Gbeta3 selectively binds G{gamma} isoforms, but at a low level even in the best of cases.

Analysis of Gbeta and G{gamma} Subunits on Nondenaturing Gels—The inference from our previous work (12) and from the data in Fig. 1, as well as from analogous experiments with mutants of conserved Asp residues in Gbeta (18), was that other constituents of the reticulocyte lysate interact with Gbeta isoforms and are involved in Gbeta subunit dimerization with G{gamma}. Nondenaturing (native) gel analysis was used to identify constituents of the reticulocyte lysate that interact with either Gbeta or G{gamma}. Interpretation of results with nondenaturing gels is more complex than with SDS-polyacrylamide gels because protein migration is determined by multiple properties, including molecular mass, pI, and shape. However, native gels preserve many interactions that would be disrupted with SDS and/or beta-mercaptoethanol, and so migration is also affected by formation of protein complexes. Taking all of these factors into account, globular proteins (or complexes) of similar pI will still travel on nondenaturing gels according to their molecular mass (or log molecular mass). This was (approximately) true, for example, for standards used in the characterizing the mobility of Gbeta, G{gamma}, and Gbeta{gamma} dimers (Fig. 2), where we used a set of globular proteins with pI values of 5.4–6.8. The Gbeta{gamma} dimer deviates somewhat, but not drastically, from being a globular protein (14), and it has a pI dominated by the Gbeta component predicted to be between 5.6 and 6.0 for the different isoforms. Thus, our expectation was that the dimer, and perhaps the folded Gbeta protein alone, would travel with an apparent molecular mass on nondenaturing gels only slightly greater than its predicted 45 kDa. On the other hand, G{gamma} alone is small (8 kDa) and of heterogeneous pI among its isoforms and appears to have defined structure based primarily upon its association with Gbeta (14). Thus, the behavior of G{gamma} on nondenaturing gels was less predictable.

When in vitro translated, 35S-labeled G{gamma} isoforms were analyzed on native gels (Fig. 2A), they did not travel with similar mobility, as they would on SDS-polyacrylamide gels. In fact, they appeared to segregate primarily according to their pI, as shown in the accompanying graph (Fig. 2B). Those G{gamma} subunits with a pI near 7, i.e. G{gamma}2, G{gamma}3, G{gamma}4, G{gamma}8c, G{gamma}8olf, and G{gamma}10, migrated in the middle of the gel. Those with a pI <7, i.e. G{gamma}1, G{gamma}11, and G{gamma}13, migrated more rapidly on the gel. And those with a pI >7, i.e. G{gamma}5, G{gamma}7, and G{gamma}12, either did not enter the gel (i.e. G{gamma}5) or migrated near the top. There were also two non-specific bands that were present in all of the lanes, including the pcDNA control lane (Fig. 2A, arrows). For some G{gamma} isoforms, for example, G{gamma}8c, G{gamma}8o, and G{gamma}11, two bands were seen. G{gamma} subunits have post-translational modifications, including by farnesylation or geranylgeranylation at the C terminus and by N-terminal processing that can include removal of methionine and/or acetylation of the N terminus. Variable post-translational modification at these sites, as we have described recently for brain G{gamma} subunits (32), may affect the hydrophobicity and/or shape of the G{gamma} subunits such that their migration on nondenaturing gels is affected.

The behavior of Gbeta isoforms on native gels (Fig. 2C) was quite different from that of the G{gamma} isoforms (Fig. 2A). For all the in vitro transcribed Gbeta isoforms, 35S-labeled protein accumulated in two primary locations (Fig. 2C): at the top of the native gel, presumably as denatured, aggregated protein of a size too great to enter the gel, and as a sharp, well delineated band at >669 kDa (band a). This band is specific to translation of Gbeta and was not found in the pcDNA control or after translation of G{gamma} (Fig. 2C). There was also some labeled material near the 67-kDa marker that was diffuse for Gbeta1, Gbeta2, Gbeta3, and Gbeta4; sharper for Gbeta5 (band f); and prominent but diffuse for Gbeta5L (band e). This material migrated at a molecular mass slightly larger than expected for Gbeta subunits, perhaps representing incompletely folded protein. There were also two non-specific bands (bands b and d), also seen with G{gamma} (Fig. 2A), and a band of variable intensity at ~140 kDa (band c), which might be a dimer of the material at ~60–70 kDa or Gbeta complexed with other specific proteins. Most interesting in these samples are the bands at >669 kDa. Because Gbeta was the only significantly labeled protein on this gel (see Fig. 1A, for example), the best explanation for band a is that it is Gbeta migrating at a very high molecular mass in association with a multiprotein complex. The fact that this is a sharp band supports this idea because denatured and/or aggregated protein either would not enter the gel (as seen at the top) or would have heterogeneous mobility and produce a protein smear, as would incompletely or randomly folded protein.


Figure 2
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  		FIGURE 2.
Separation of G{gamma} and Gbeta subunits on native (nondenaturing) gels. A, native (nondenaturing) gel of G{gamma} subunits. G{gamma} subunits were expressed in the reticulocyte lysate and labeled with [35S]methionine. Samples were run on a nondenaturing 4–15% gradient gel, and the gel was dried and autoradiographed. Arrowheads indicate positions of G{gamma}; arrows indicate nonspecific bands present in all samples. B, pI values for G{gamma}. The pI values for the G{gamma} subunits were predicted using the Compute pI/Mw tool available on the ExPASy proteomics server (available at us.expasy.org/tools/pi_tool.html). C, native (nondenaturing) gel analysis of Gbeta subunits. Gbeta subunits or FLAG-G{gamma}3 was expressed in the reticulocyte lysate and labeled with [35S]methionine. Samples were run on a nondenaturing 4–15% gradient gel, and the gel was dried and autoradiographed. Band a, Gbeta of high apparent molecular mass; band b, nonspecific band present in all samples; band c, band seen with Gbeta1, Gbeta2, Gbeta3, and Gbeta4; band d, nonspecific band present all samples; band e, band specific to Gbeta5L; band f, band specific to Gbeta5. The gray arrow indicates the top of the gel.

 


Figure 3
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  		FIGURE 3.
Behavior of CCT/TRiC on native (nondenaturing) gels and its co-immunoprecipitation with Gbeta. A, immunoblot of CCT/TRiC on a nondenaturing gel. Gbeta1 and FLAG-G{gamma}3 were synthesized with unlabeled methionine and combined to allow dimer formation. Samples were run on an 8–16% nondenaturing gel, transferred, and immunoblotted (IB) with antibody to the CCT/TRiC subunit TCP-1{alpha}. Lane 1, Gbeta1; lane 2, FLAG-G{gamma}3; lane 3, Gbeta1 and FLAG-G{gamma}3 at 2:1; lane 4, Gbeta1 and FLAG-G{gamma}3 at 2:3; lane 5, pcDNA3. The approximate positions of molecular mass standards (in kilodaltons) are shown on the left. Note that the position of TCP-1{alpha} on the gel, traveling slightly slower than the 669-kDa standard, is similar to the position of the Gbeta band labeled a in Fig. 2C. B, Gbeta1 co-immunoprecipitates with CCT/TRiC. Unlabeled FLAG-Gbeta1 and unlabeled FLAG-G{gamma}3 were separately expressed and immunoprecipitated (IP) using anti-FLAG beads. Duplicate samples were separated by SDS-PAGE on an 8–16% gel, transferred, and immunoblotted with rat monoclonal anti-TCP-1{alpha} antibody (left) or rabbit anti-Gbeta subunit antibody BC1 (right). Lanes 1, FLAG-Gbeta1; lanes 2, FLAG-G{gamma}3; lanes 3, pcDNA3. The approximate positions of molecular mass standards (in kilodaltons) are indicated on the right. The secondary antibody for TCP-1{alpha} is anti-rat, whereas the secondary antibody for BC1 is anti-rabbit. (The anti-rat secondary antibody used in the TCP-1{alpha} immunoblot recognized mouse heavy and light chains (indicated with gray arrows on the left) that co-eluted from the anti-FLAG beads (containing a mouse monoclonal antibody).) Note that TCP-1{alpha} co-immunoprecipitated only with FLAG-Gbeta1 (lanes 1), but not with FLAG-G{gamma}3 (lanes 2) or with anti-FLAG beads from a mock translation (lanes 3).

 
Characterization of the Interaction of Gbeta with the Chaperonin CCT/TRiC Complex—The apparent size of the interacting partner on native gels and previous reports that some WD repeat proteins interact with or are folded by CCT/TRiC (19, 25, 27) suggested that one candidate for the binding partner affecting the mobility of Gbeta subunits on native gels is the chaperonin CCT/TRiC complex. The intact CCT/TRiC protein, with an approximate molecular mass of ~800 kDa, migrates very slowly on a nondenaturing gel (28, 33). This is shown in Fig. 3A, where samples of the reticulocyte lysate with or without expressed proteins (FLAG-Gbeta1 and FLAG-G{gamma}3) were run on a native gel and immunoblotted with an antibody specific for the {alpha}-subunit of CCT/TRiC (TCP-1{alpha}). All these samples contained a high molecular mass band recognized by anti-TCP-1{alpha} antibody at a high molecular mass similar to that seen in the Gbeta subunit lanes (Fig. 2). Thus, Gbeta synthesized in the reticulocyte lysate migrates on native gels in a region also containing a high molecular mass complex of the CCT/TRiC subunits. To more definitively evaluate whether Gbeta and CCT/TRiC co-migrate on nondenaturing gels, we stained the immunoblot with antibodies tagged with infrared dyes that are amenable to double staining of immunoblots (Fig. 4). FLAG-Gbeta1, FLAG-Gbeta2, or the pcDNA control was translated in the reticulocyte lysate and then separated on either nondenaturing (Fig. 4A) or denaturing (SDS) (Fig. 4B) gels. Proteins transferred to nitrocellulose were blotted simultaneously with anti-TCP-1{zeta} and anti-FLAG antibodies and visualized with secondary antibodies tagged with different infrared-emitting dyes (green for TCP-1{zeta} and red for FLAG in Fig. 4). On SDS-polyacrylamide gels, Gbeta and TCP-1{zeta} traveled independently of one another and at their expected monomer molecular masses (Fig. 4B). On nondenaturing gels, however, Gbeta and TCP-1{zeta} traveled at a high molecular mass that could be co-localized as a merged signal of the two tagged antibodies. These data demonstrate that Gbeta and TCP-1{zeta} co-migrate on nondenaturing gels, suggesting their association in a complex.


Figure 4
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  		FIGURE 4.
Co-migration of TCP-1{alpha} and FLAG-Gbeta on nondenaturing gels. FLAG-Gbeta1 and FLAG-Gbeta2 were individually synthesized with rabbit reticulocyte lysate in the presence of unlabeled methionine. Samples of the translated proteins or translation mixture incubated with pcDNA3 with no insert (control (C)) were run on 4–20% gradient gels under native (A) and denaturing (SDS) (B) conditions, transferred, and immunoblotted simultaneously with anti-FLAG (rabbit) and anti-TCP-1{zeta} (goat) antibodies. Secondary antibodies were tagged with infrared-emitting labels: secondary antibody to FLAG (donkey anti-rabbit IgG) emitting at 700 nm and colored red and secondary antibody to TCP-1{alpha} (donkey anti-goat IgG) emitting at 800 nm and colored green. Immunoblots were scanned in an Odyssey infrared imager (LI-COR). In each case, the identical immunoblot is shown with its red-emitting signal (anti-FLAG antibody) and its green-emitting signal (anti-TCP-1{zeta} antibody) and as the simultaneous signal, with yellow indicating co-localization of the two signals. The approximate positions of molecular mass markers (in kilodaltons) are shown on the left. Note that Gbeta and TCP-1{zeta} traveled at their subunit molecular masses in B, but co-migrated at high apparent molecular mass in A, an apparent molecular mass that is compatible with that of the intact CCT/TRiC complex.

 


Figure 5
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  		FIGURE 5.
Immunoprecipitation of CCT/TRiC subunit isoforms with FLAG-Gbeta synthesized in reticulocyte lysates. A, Coomassie Blue-stained gel of FLAG-immunoprecipitated samples of the reticulocyte lysate translated with pcDNA3 containing Gbeta1 (+) or the empty pcDNA3 vector (–). Antibody heavy chain (Ab(HC)) and light chain (Ab(LC)) bands common to both lanes are labeled on the right. Bands labeled TCP are unique to the Gbeta1 lane. The assignment of these bands as TCP subunits is based upon the data in Table 1. The position of FLAG-Gbeta1 is indicated on the right. This was a weak band commensurate with the low level of protein produced in the reticulocyte lysate (estimated to be 50–100 ng in this sample). Stoichiometric amounts of CCT/TRiC to Gbeta1 should generate a ratio of TCP bands to a FLAG-Gbeta band of ~30:1 based upon equivalent staining with Coomassie Blue. B, enlarged image of unique bands labeled TCP in the Coomassie Blue-stained lane containing FLAG-Gbeta. The approximate positions of the five gel slices analyzed by MALDI-TOF/TOF after trypsin digestion are labeled 1–5, the data from which are summarized in Table 1. C, Coomassie Blue-stained gel of 1 µl of rabbit reticulocyte lysate run between lanes containing molecular mass standards and demonstrating the specificity of the immunoprecipitation shown in A.

 
To determine whether Gbeta and the CCT/TRiC subunits are actually physically associated, we evaluated whether the subunits co-immunoprecipitate. Unlabeled FLAG-Gbeta1, FLAG-G{gamma}3, or the pcDNA control was expressed in lysate and subjected to immunoprecipitation with anti-FLAG beads (Fig. 3B). Only the FLAG-Gbeta1 immunoprecipitates contained TCP-1{alpha} immunoreactivity. Notably, the FLAG-G{gamma}3 and pcDNA control samples contained only the heavy and light chains from the anti-FLAG antibody and nothing in the position of TCP-1{alpha} or Gbeta. Thus, FLAG-Gbeta, but not FLAG-G{gamma}, binds to TCP-1{alpha}. To more generally evaluate the specificity of association of CCT/TRiC with Gbeta and to confirm that the immunoreactivity observed in Fig. 3 was in fact TCP-1{alpha}, we stained SDS gels of immunoprecipitates with Coomassie Blue (Fig. 5A). In comparing control (pcDNA3) and FLAG-beta1 samples, we observed several protein bands between 50 and 60 kDa associated with the FLAG-beta1 sample (Fig. 5B). The specificity of this complex of proteins for association with FLAG-Gbeta was striking given that reticulocyte lysate is essentially intact cytosol containing a large amount of hemoglobin (Fig. 5C). To identify the constituents of this complex, we used mass spectrometric (MS) peptide mapping and MS/MS sequencing of trypsin digests of the bands excised from the gel (Table 1). The results were analyzed using the Mascot search algorithm (31) to evaluate which proteins in a library of 163,000 rodent proteins (NCBI Protein Database) could account for the MS fragments and MS/MS-sequenced peptides identified. For all five bands, the only proteins with significant protein scores were TCP subunits or predicted proteins from genome data bases that are TCP homologs. For the highest scoring matches, we obtained both MS fingerprinting data and MS/MS sequencing of multiple peptides. The only peptide identified in these experiments that was not a TCP subunit or homolog was an IgG heavy chain fragment. This is an expected contaminant in this molecular mass range from the immunoprecipitation procedure.


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  		TABLE 1
MS fingerprinting and MS/MS sequencing identification of proteins that immunoprecipitate with FLAG-Gbeta synthesized in reticulocyte lysates

FLAG-Gbeta 1 was synthesized in the reticulocyte lysate and immunoprecipitated with anti-FLAG antibody. Control samples were translated in the presence of pcDNA3 lacking a functional insert. A representative Coomassie Blue-stained gel is shown in Fig. 5. The bands labeled 1-5 in Fig. 5B were excised from the gel with a scalpel and digested with trypsin as described under "Materials and Methods." Extracts from the trypsin digestion were analyzed by MALDI-TOF/TOF on an Applied Biosystems 4700 proteomics analyzer. The 10 most intense peaks in each spectrum were sequenced by MS/MS. Data were analyzed using ABI GPS Explorer 3.5 software implementing the Mascot search algorithm (31).

 
CCT/TRiC is thought to be a dynamic complex in cells, existing both as an intact complex and as individual subunits (34). To confirm the interaction of Gbeta with CCT/TRiC and to evaluate whether different Gbeta subunits interact selectively with TCP subunits or with the CCT/TRiC complex, we immunoprecipitated FLAG-Gbeta constructs from the reticulocyte lysate and immunoblotted for all eight TCP subunits (Fig. 6). We characterized the level of each TCP isoform in the reticulocyte lysate as well as any TCP immunoprecipitated from a mock translation with pcDNA3 or with translated FLAG-G{gamma}2 as controls (Fig. 6A). All of the anti-TCP isoform antibodies recognized bands in the lysate, except anti-TCP-1{delta} antibody (data not shown). TCP-1{delta} was probably present in the lysate, but the antibody (raised against a human sequence) may not recognize rabbit protein. Nevertheless, seven of the eight isoforms were readily identifiable. TCP isoforms were present in immunoprecipitates for all of the Gbeta constructs, but none of the seven isoforms assayed was present in the pcDNA3 mock translation or in the immunoprecipitates of FLAG-G{gamma}2 (Fig. 6). Samples (Fig. 6A) were normalized for FLAG-Gbeta expression using anti-FLAG immunoblotting, and the level of each TCP isoform was compared for each of the Gbeta isoforms (Fig. 6B). These results indicate that Gbeta, but not G{gamma}, associates with the CCT/TRiC complex after synthesis and that Gbeta associates with the complex and not just with isolated TCP subunits. This is a compelling explanation for the behavior of the Gbeta isoforms on native gels (Fig. 2). In addition, the analysis of the relative amounts of TCP isoforms recovered with each Gbeta subunit varied selectively with the Gbeta isoform in the order of Gbeta5 < Gbeta3s < Gbeta3 < Gbeta2 ≤ Gbeta1 ≤ Gbeta4 (Fig. 6, B and C). Interestingly, this corresponds closely to our recent results describing the efficiency of specific Gbeta isoforms forming functional Gbeta{gamma} dimers (12) and suggests that there is a relationship between the Gbeta interaction with CCT/TRiC and dimer formation.

Assay of Gbeta{gamma} Dimer Assembly on a Nondenaturing Gel—The results with native gels (Fig. 2) suggested an approach for studying Gbeta{gamma} dimer formation, including the role of intermediates in this process, such as Gbeta association with CCT/TRiC. To further investigate any relationship between the various Gbeta and G{gamma} bands observed on native gels (Fig. 2) and the mobility of Gbeta{gamma} dimers, 35S-labeled Gbeta was combined with unlabeled G{gamma}1 or G{gamma}3, or unlabeled Gbeta was combined with 35S-labeled G{gamma}1 or G{gamma}3; and after dimerization, samples were run on a nondenaturing gel (Fig. 7). Lanes 1–6 correspond to labeled Gbeta1, and lanes 7–14 correspond to labeled FLAG-G{gamma}. Gbeta1 without G{gamma} (lanes 1 and 2) had one specific band near the top of the gel at ~669 kDa. This band was seen only in samples containing labeled Gbeta1 (lanes 1–6) and not in samples containing labeled G{gamma} (lanes 7–14). New bands, not seen in the Gbeta- or G{gamma}-only lanes, appeared in all lanes containing labeled Gbeta1 with FLAG-G{gamma}1 or with FLAG-G{gamma}3 (lanes 3–6); these bands represent the dimers Gbeta1/FLAG-G{gamma}1 and Gbeta1/FLAG-G{gamma}3. The apparent size difference observed for the two dimers can be explained by the pI differences in the two G{gamma} subunits (see also Fig. 2B). The conclusion that the new bands seen with 35S-labeled Gbeta and unlabeled G{gamma} in lanes 3–6 are Gbeta{gamma} dimers is corroborated by the observation that bands at the identical positions were seen for Gbeta1/FLAG-G{gamma}1 and Gbeta1/FLAG-G{gamma}3, respectively, in lanes 11–14, where the labeled partner was G{gamma} instead of Gbeta1. The bands corresponding to Gbeta{gamma} dimers were absent in the lanes containing labeled G{gamma} without Gbeta (lanes 7 and 8 for FLAG-G{gamma}1 and lanes 9 and 10 for FLAG-G{gamma}3) (immediately under the faster migrating nonspecific band) (Fig. 7B). Fig. 7B shows that the bands for FLAG-G{gamma}3 and the Gbeta1/FLAG-G{gamma}3 dimer are different. Significantly, 35S-labeled G{gamma} was not seen in association with the CCT/TRiC band when expressed either alone or with Gbeta and forming a Gbeta{gamma} dimer (lanes 7–14). These experiments demonstrate that Gbeta{gamma} dimer formation can be observed on native gels and provide additional evidence for the stability of these dimers because they remain associated during electrophoresis.


Figure 6
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  		FIGURE 6.
Interaction of Gbeta isoforms with the CCT/TRiC complex. A, FLAG-Gbeta isoforms were translated in the reticulocyte lysate with unlabeled methionine and immunoprecipitated (IP) with anti-FLAG beads. The immunoprecipitates were run on an SDS-polyacrylamide gel, transferred, and separately immunoblotted (IB) with antibodies specific for the chaperonin TCP subunits {alpha}, beta, {gamma}, {epsilon}, {zeta}, {eta}, and {theta}. Each blot was subsequently blotted with anti-FLAG antibody to visualize FLAG-Gbeta. For the TCP immunoblots, only the region of the gels corresponding to 50–65 kDa is shown; the expected molecular masses of the TCP subunits are all ~60 kDa. For the FLAG immunoblots, only the region of the blot corresponding to 30–50 kDa is shown. Arrows indicate the positions of the individual TCP subunits. B, immunoblots for each chaperonin subunit (TCP) and its corresponding anti-FLAG blot were imaged with a Bio-Rad Fluor-S MAX MultiImager, and the signal was quantitated. The data were normalized to reflect the relative amount of a given chaperonin subunit for the amount of each Gbeta subunit present. Each experiment was done a minimum of three times, and the normalized data were averaged. Data are graphed as mean ± S.E. C, shown is a summary of the Gbeta isoform interaction with the CCT/TRiC complex.

 
To evaluate the relationship between Gbeta and G{gamma} synthesis and dimer assembly, Gbeta1, either alone or in combination with FLAG-G{gamma}3 cDNA, was expressed in lysate with [35S]methionine. Over the course of 2 h, samples were removed at the time points (minutes) shown in Fig. 8. Fig. 8A shows the time course of Gbeta1 subunit synthesis without FLAG-G{gamma}1.Gbeta1 could be seen after 10 min, migrating at the high molecular mass of the CCT/TRiC complex. When cDNAs for both subunits were included (Fig. 8B), FLAG-G{gamma}1 appeared sooner than Gbeta1, as early as 4 min, and the Gbeta1/CCT/TRiC band appeared at 10 min, as for Gbeta1 alone (Fig. 8A). The presence of G{gamma} cDNA or G{gamma} protein (Fig. 8B) did not affect the timing of synthesis of Gbeta, although it did affect its distribution by decreasing the amount of both smeared radioactivity on the gel (presumably mis-folded Gbeta protein) and radioactivity at the top of the gel (aggregated protein). The Gbeta1/FLAG-G{gamma}1 dimer appeared by 30 min, after the appearance of both FLAG-G{gamma}1 and the Gbeta1/CCT/TRiC complex.

Role of CCT/TRiC in Gbeta{gamma} Dimer Assembly—The results shown in Fig. 8 indicate that dimer formation follows Gbeta interaction with CCT/TRiC and is compatible with a role of CCT/TRiC in Gbeta{gamma} dimer assembly. CCT/TRiC has an integral ATPase activity that has been linked to closing of the CCT/TRiC folding chamber and facilitating protein folding as well as releasing the folded substrate from the complex (21, 22, 26). This ATPase activity has also been associated with complex formation in the assembly of specific multimeric structures (35–37). The functions of this ATPase activity can be blocked by addition of adenine nucleotide analogs such as ATP{gamma}S and ADPbetaS or by chelation of Mg2+. In addition, the "closed" state of the CCT/TRiC complex is thought to correspond to ADP·Pi, the mid-hydrolysis state of ATP. ADP·Pi can be mimicked by ADP and by Al(NO3)3, MgCl2, and NaF, whereby the fluoride coordinates with the aluminum in a manner that mimics the {gamma}-phosphate of ATP. Previous studies have not evaluated whether Gbeta{gamma} dimer formation requires ATP independently of Gbeta or G{gamma} synthesis. One prediction of a role of CCT/TRiC in dimer formation would be that this process is ATP-dependent; if it were not, this would suggest that the role of CCT/TRiC is primarily in the folding of Gbeta, rather than participating in dimer formation directly. We therefore tested whether Gbeta{gamma} dimer formation is ATP-dependent.


Figure 7
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  		FIGURE 7.
Gbeta{gamma} dimer formation assayed on a nondenaturing gel. To determine whether nondenaturing gels could be used to monitor Gbeta{gamma} dimer formation, Gbeta or G{gamma} was independently labeled with [35S]methionine, and their mobility on 4–15% nondenaturing gels was evaluated with or without preincubation with the non-radioactively labeled complementary subunit. A: lanes 1–6, Gbeta1 was synthesized in the presence of [35S]methionine and combined with G{gamma}1 or G{gamma}3 that was synthesized in the presence of unlabeled methionine. 35S-Labeled Gbeta1 alone (lanes 1 and 2) traveled primarily at high molecular mass. If preincubated with either unlabeled G{gamma}1 (lanes 3 and 4) or unlabeled G{gamma}3 (lanes 5 and 6), 35S-labeled Gbeta1 had increased mobility dependent on with which G{gamma} isoform it was preincubated (arrows). Lanes 7–14, G{gamma}1 (lanes 7 and 8) or G{gamma}3 (lanes 9 and 10) was synthesized in the presence [35S]methionine; their mobility on the nondenaturing gel was isoform-specific. When unlabeled Gbeta1 was preincubated with labeled G{gamma}1 (lanes 11 and 12) or with labeled G{gamma}3 (lanes 13 and 14), a new band of altered mobility was observed at a similar position as observed for the 35S-labeled Gbeta1 samples in the presence of unlabeled G{gamma} (lanes 3–6). Dimer-specific bands are indicated by arrows. B: an enlargement of the area in A shows G{gamma}3 alone and Gbeta1{gamma}3-specific bands (arrows).

 
Gbeta1, Gbeta2, FLAG-G{gamma}1, and FLAG-G{gamma}3 were individually expressed with unlabeled methionine or [35S]methionine for 2 h at 37 °C and then incubated in combinations containing one unlabeled and one labeled subunit. From the time course in Fig. 8, it is apparent that any requirement for folding of Gbeta, either by CCT/TRiC or other chaperones, can be completed in 60 min or less because the Gbeta{gamma} dimer will have already appeared by then. In addition, in other studies, we found that productive dimer formation did not proceed beyond 2 h (data not shown). Before combining the subunits, ATP; Al(NO3)3, MgCl2, and NaF; ATP{gamma}S; or ADPbetaS was added to the expressed Gbeta subunit (Fig. 9). Fig. 9A shows control results with 5 mM ATP. Incubation of FLAG-G{gamma}3 with either Gbeta1 or Gbeta2 resulted in generation of new 35S-labeled Gbeta bands and a slight change in the mobility of 35S-labeled FLAG-G{gamma}3. Incubation of FLAG-G{gamma}1 with Gbeta1 resulted in a new band that could be labeled with either 35S-labeled Gbeta or 35S-labeled FLAG-G{gamma}1. In contrast and as expected from previous work (see "Discussion" in Ref. 12), FLAG-G{gamma}1 did not form a dimer with Gbeta2. During dimerization in the presence of Al(NO3)3, MgCl2, and NaF (Fig. 9B); ATP{gamma}S (Fig. 9C); or ADPbetaS (Fig. 9D), the radioactivity associated with the dimer was abolished or greatly reduced. Conversely, the amount of 35S-labeled Gbeta migrating with CCT/TRiC was increased under these same conditions. This suggests that the Gbeta involved in dimer formation came from that associated with CCT/TRiC and that this complex is the relevant target for the effect of ATP analogs on the dimerization process. The nucleotide analogs did not promote G{gamma} association with CCT/TRiC, and Gbeta2{gamma}1, a dimer that does not normally occur, was not seen either as dimer or as an intermediate of that dimer. Additional experiments showed that chelation of Mg2+ with EDTA also abolished dimer formation (data not shown).


Figure 8
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  		FIGURE 8.
Time course of dimer formation between Gbeta1 and FLAG-G{gamma}1 on a nondenaturing gel. 35S-Labeled Gbeta1 was expressed in the reticulocyte lysate alone (A) or with FLAG-G{gamma}1 to allow dimer formation (B). Samples were taken at the times indicated (minutes after addition of both cDNAs) and run on a native gel. A, Gbeta1 at apparent high molecular mass (black arrow) complexed with CCT/TRiC was first seen at ~10 min and increased in amount for times up to 120 min. Increasing background radioactivity with time suggests accumulation of randomly folded and/or denatured protein with variable mobility. B, FLAG-G{gamma}1 (gray arrow) was seen at ~4 min. Gbeta{gamma}1 as a complex with CCT/TRiC (upper black arrow) was seen at 10 min, as when synthesized alone (as in A). The Gbeta1/FLAG-G{gamma}1 dimer (lower black arrow) appeared by 30 min, after the appearance of both FLAG-G{gamma}1 and the high molecular mass Gbeta1, and continued to accumulate at times up to 120 min.

 
The data in Fig. 9 suggest that CCT/TRiC mediates Gbeta{gamma} dimer formation via an ATP-dependent process that involves an interaction with Gbeta, but not G{gamma}. We tested this conclusion by evaluating whether addition of G{gamma} affects Gbeta association with CCT/TRiC. FLAG-Gbeta1 and G{gamma}2 were independently synthesized with unlabeled methionine, and then either excess lysate (5-fold) containing G{gamma}2 or mock-translated lysate (with pcDNA3) was added to lysate containing FLAG-Gbeta1. After 90 min, FLAG-Gbeta1 was immunoprecipitated, and samples were blotted for TCP-1{gamma} and FLAG-Gbeta1 (Fig. 10). In the absence of G{gamma}2, TCP-1{gamma} immunostaining was more prominent than FLAG-Gbeta1 staining (Fig. 10B). Immunostaining of FLAG-Gbeta1 in these samples was about equal in intensity with the control (Fig. 10A) and represents immunoprecipitation of 20–30% of the synthesized FLAG protein. This is compatible with the data in Fig. 8, suggesting that, in the absence of G{gamma}, Gbeta protein tends to be misfolded or aggregated and less accessible to anti-FLAG antibody. If synthesized FLAG-Gbeta1 was incubated with lysate containing G{gamma}2, the amount of FLAG-Gbeta1 recovered in the immunoprecipitation increased to 70–80% of the total (Fig. 10, compare A and B), whereas the amount of TCP-1{gamma} associated with FLAG-Gbeta1 decreased both in amount relative to FLAG-Gbeta1 and in absolute amount. This result, particularly the absolute decrease in the amount of TCP-1{gamma} associated with FLAG-Gbeta1, implies that G{gamma} and CCT/TRiC compete for binding of Gbeta during the process of Gbeta{gamma} dimer formation.


Figure 9
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  		FIGURE 9.
Effect of adenosine nucleotide analogs on Gbeta{gamma} dimer assembly. Gbeta1, Gbeta2, FLAG-G{gamma}1, or FLAG-G{gamma}3 was expressed in the reticulocyte lysate with [35S]methionine and then incubated in the indicated combinations at 37 °C for 2 h in the presence of 5 mM ATP (A); 50 µM Al(NO3)3, 5 mM MgCl2, and 10 mM NaF (AMF; B); 5 mM ATP{gamma}S(C); or 5 mM ADPbetaS(D). Samples were run on a nondenaturing gel, and the dried gel was exposed to a PhosphorImager screen for visualization. The Gbeta, G{gamma}, and Gbeta{gamma} combinations are indicated at the tops of the gels. Gbeta{gamma} bands are marked in A and are eliminated or greatly reduced in B–D.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data reported here indicate that Gbeta isoforms bind the chaperonin CCT/TRiC complex, which is known to be involved in protein folding (19, 21, 38) and to be a mediator of assembly for selected protein complexes (35, 36). Gbeta co-migrated with CCT/TRiC on nondenaturing gels (Figs. 2, 3, 4), and FLAG-Gbeta co-immunoprecipitated CCT/TRiC (Figs. 2, 5, 6, and 10 and Table 1). These data indicate that Gbeta associates with the CCT/TRiC complex and not just with individual TCP subunits (Figs. 2 and 6). In contrast, CCT/TRiC did not associate with G{gamma} isoforms either on nondenaturing gels (Fig. 2) or by co-immunoprecipitation (Fig. 6). Most chaperones are major cellular constituents found in all cells. Despite this, analysis by MS fingerprinting and MS/MS sequencing (Table 1) of FLAG-Gbeta immunoprecipitated from Coomassie Blue-stained gels (Fig. 5) identified only TCP subunits. This observation was confirmed by immunoblotting for other major chaperone proteins, including Hsp40, Hsp70, and Hsp90.3 This does not negate the involvement of other proteins or cofactors, but it does suggest a primary role for CCT/TRiC in the post-translational events leading to dimer formation.

We report here substantial evidence for the role of CCT/TRiC in the folding of Gbeta and as a major determinant or intermediate of Gbeta{gamma} dimer formation. These include the following. 1) Gbeta specifically binds CCT/TRiC (Figs. 5 and 6), a complex involved in protein folding (19, 21, 38) and protein complex formation (35, 36), shortly after its synthesis (Fig. 8); there is a lag of several minutes before dimer formation, as would be expected if folding of Gbeta on CCT/TRiC is a requirement for production of functional Gbeta.2) Gbeta association with CCT/TRiC is isoform-specific (Fig. 6), and the specificity of these interactions correlates closely with the ability of different Gbeta isoforms to form dimers in the same rabbit reticulocyte lysate system as described here (Fig. 1) and previously (12). The selectivity of these interactions argues against nonspecific association of unfolded or misfolded, newly synthesized Gbeta with CCT/TRiC and is consistent with observations that the protein substrates of this complex interact with CCT/TRiC through specific sequences (19, 23, 24). 3) We show here for the first time that Gbeta{gamma} dimer formation is ATP-dependent and that this is a requirement separate from synthesis or folding of either Gbeta or G{gamma} (Fig. 9). This is both a prediction and a test of the involvement of CCT/TRiC in dimer formation because nearly all of its activities, including folding, release of folded peptide, and complex formation, have been shown to be ATP-dependent (21, 22, 26, 35–37). That the target of this inhibition is CCT/TRiC is supported strongly by the observation that inhibition of ATPase activity leads to increased association of labeled Gbeta association with the CCT/TRiC complex (Fig. 9). 4) Addition of G{gamma} to previously synthesized Gbeta under conditions allowing dimer formation (Fig. 1) reduces Gbeta association with CCT/TRiC (Fig. 10), indicating that Gbeta bound to CCT/TRiC is capable of functionally interacting with G{gamma} and that exposure of the complex to G{gamma} results in release of Gbeta from CCT/TRiC.

Our results are summarized in the model shown in Fig. 11. Shortly after synthesis, Gbeta associates with CCT/TRiC. This would most likely be mediated through specific sites of interaction on Gbeta, as is found for most well characterized substrates (19, 21, 38), and may require partial folding of the protein, perhaps mediated by other chaperones. The specificity of this interaction would explain the variable association of different Gbeta isoforms with CCT/TRiC (Fig. 6). ATP-dependent events would then be required to mediate folding of Gbeta to a state competent to bind G{gamma} subunits. There is evidence that, for many substrates, the folding process on CCT/TRiC proceeds through rounds of binding and ATP-dependent release, ultimately leading to properly folded native protein (39). Release of properly folded protein in the presence of G{gamma} subunits would allow binding of the two partners and prevent rebinding of Gbeta (as a Gbeta{gamma} dimer) to CCT/TRiC. This is essentially what we observed, i.e. when G{gamma} is added to presynthesized Gbeta, it results in dissociation of Gbeta from CCT/TRiC (Fig. 10). This result is comparable with three other cases in which CCT/TRiC mediates complex formation: association of the von Hippel-Lindau protein with its partner elongin B/C (33, 35), complex formation between histone deacetylase-3 and its binding partner SMRT (36), and formation of a cyclin E·CDK2 complex (37). Although the details differ somewhat between these various reactions, in all three cases, one component is folded on CCT/TRiC and is removed from the complex in an ATP-dependent reaction by its binding partner, which itself does not appear to interact with the CCT/TRiC complex.


Figure 10
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  		FIGURE 10.
Effect of G{gamma}2 on the association of TCP-1{gamma} with FLAG-Gbeta1. FLAG-Gbeta1 and G{gamma}2 were separately synthesized in rabbit reticulocyte lysate for 2 h at 30°C. Duplicate samples of FLAG-Gbeta1 (10 µl of lysate) were then combined with either the control (pcDNA3) or G{gamma}2-containing lysate (50 µl) and incubated for 90 min to allow dimer formation. Samples were immunoprecipitated with anti-FLAG beads, run on a 4–20% gradient gel, transferred to nitrocellulose, and immunostained first with antibody to TCP-1{gamma} and then with antibody to FLAG. A, immunoblot of 2 µl of the starting lysate containing FLAG-Gbeta1 after sequential staining with anti-TCP-1{gamma} antibody and then anti-FLAG antibody. B, immunoblot of FLAG-immunoprecipitated (IP) samples after sequential staining with anti-TCP-1{gamma} antibody and then anti-FLAG antibody. Ab(HC) and Ab(LC), antibody heavy and light chains, respectively. C, lanes from same gels as in A and B containing 1 µl of the final incubation mixture before immunoprecipitation. Only the TCP-1{gamma} band is shown, demonstrating that equivalent samples were loaded on the gel.

 


Figure 11
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  		FIGURE 11.
Model showing the role of CCT/TRiC in the synthesis of Gbeta and in the formation of the Gbeta{gamma} dimer. Gbeta associates with CCT/TRiC shortly after synthesis. Folding of proteins by CCT/TRiC is known to be ATP-dependent, as is release of the folded protein from the complex. We have not here found evidence of association of G{gamma} with CCT/TRiC or with other proteins, but its presence either during or after Gbeta synthesis promotes release of Gbeta from CCT/TRiC and formation of the Gbeta{gamma} dimer. This process is also ATP-dependent. This model does not exclude the participation of other chaperone proteins or cofactors such as PhLP, which may be a co-chaperone or a regulator of Gbeta folding and Gbeta{gamma} assembly (20, 46).

 
This work explains a number of past observations about Gbeta folding and Gbeta{gamma} dimer assembly that include the following findings: Gbeta does not fold correctly without G{gamma} (16, 40); prokaryotes do not have the ability to assemble Gbeta{gamma} dimers (17); some in vitro systems, such as the plant wheat germ system, also lack the ability to form dimers (17); cellular requirements for dimer formation are not completely or necessarily met in in vitro assembly systems (12, 18); and there are structural requirements associated with Gbeta subunits that determine their ability to efficiently form dimers (12, 18). It has been reported that the yeast Gbeta homolog Ste4 associates with the TCP-1{alpha} subunit in a mass screening of interacting partners in yeast (19, 27). Our results extend this observation to animal cells and to an association with the CCT/TRiC complex rather than its isolated TCP subunits. Notably, our results complement the work of Humrich et al. (20), which showed that small interfering RNA to TCP-1{alpha} decreases Gbeta{gamma} levels in human embryonic kidney 293 cells. Our studies here indicate that the requirement for TCP-1{alpha} is for folding Gbeta and not for production of some other intermediate required for dimer formation. The previous experiments (20) are also important because they indicate that the mechanism we have described here using rabbit reticulocyte lysate is likely the same mechanism used by other cells.

CCT/TRiC was originally characterized in its folding of actin and tubulin (19, 21, 41, 42) and then for other proteins, including the G{alpha} subunit of transducin (39). More recently, it has been characterized in the folding of several proteins that are assembled into complexes through a CCT/TRiC-dependent mechanism (33, 35–37). The folding of these proteins, as well as any associated complex formation, can require the participation of other proteins or chaperones, and there are multiple types of co-chaperones and mechanisms associated with these processes. In addition to the requirement for the CCT/TRiC complex shown here, Hsp90 (43) and phosducin-like protein (PhLP) (20, 44–46) may participate in Gbeta folding, the regulation of its folding, or the assembly of Gbeta{gamma} dimers. In the immunoprecipitation of FLAG-Gbeta or FLAG-G{gamma}, we did not identify any associated Hsp40, Hsp70, or Hsp90,3 but such interactions may be transient or unstable. The behavior of G{gamma} subunits on native gels (Fig. 2) seems to be largely dictated by their pI and, in contrast to Gbeta, not by their interaction with other proteins. Some G{gamma} proteins do migrate as doublets on native gels (Fig. 2), which could indicate such interactions, but could also be explained by variable post-translational modification (32). Likewise, although the mobility of Gbeta isoforms on native gels can be explained by their formation of a complex with CCT/TRiC (or by accumulation of denatured, aggregated protein on the top of the gel as in Fig. 2), there are other bands, varying in position or intensity, associated with the different isoforms. These could represent complexes with other proteins (not CCT/TRiC) involved in folding or dimer assembly.

The Gbeta-binding protein PhLP has recently been reported to have a role in Gbeta synthesis and Gbeta{gamma} dimer assembly (20, 46). One previous study indicated that PhLP binds both Gbeta and CCT/TRiC and that small interfering RNA knockdown of PhLP decreases Gbeta synthesis and Gbeta{gamma} dimer assembly (46). That work led to the novel conclusion that PhLP is a molecular chaperone for Gbeta{gamma} dimer assembly. In contrast, another study suggested that PhLP overexpression negatively regulates Gbeta synthesis and Gbeta{gamma} dimer assembly by an inferred interaction with CCT/TRiC, based upon small interfering RNA knockdown of TCP-1{alpha} (a CCT/TRiC subunit), decreasing Gbeta/Gbeta{gamma} production (20). It is not entirely clear how to reconcile those two studies with one another. It seems probable that PhLP is one of the components (co-chaperones) of the CCT/TRiC complex involved in folding Gbeta and assembling the Gbeta{gamma} dimer. One possibility, perhaps compatible with both previous studies (20, 46), is that different splice variants or phosphorylated forms of PhLP reciprocally regulate these processes. It will be particularly important to work out the sequence of events involved and the relationship of PhLP to the structure of the CCT/TRiC complex. PhLP may be a permanent component of a subset of CCT/TRiC complexes, specifically targeting them to Gbeta, or it may be a transient component interacting with Gbeta or Gbeta{gamma} at a specific stage of folding and assembly. It could be an active participant in the folding and dimer assembly process, or it could be a regulatory component. Whatever these roles are, our data indicate that the CCT/TRiC complex provides the scaffold for the events mediated by PhLP and that CCT/TRiC itself must be an active participant in the processes because PhLP is not know to possess ATPase activity, which appears to be an integral part of the dimer assembly process.

Recent studies suggest that G protein subunit isoforms extensively regulate each other's expression in cells at both the protein and mRNA levels (47). Gbeta{gamma} dimer expression is regulated both by controlled proteolysis through the proteasome (20, 48, 49) and possibly by regulation of dimer formation (20, 44). Various phosducin-like proteins have been shown to be either negative regulators of CCT/TRiC function (44, 45), suppressing Gbeta{gamma} dimer expression (20), or co-chaperones in the folding of Gbeta (46). The results reported here and the model in Fig. 11 provide a mechanism to explain these observations and suggest possible associated physiological consequences. In contrast to the other known protein dimers assembled by CCT/TRiC, Gbeta{gamma} dimers represent a combinatorially complex family of proteins with at least 60 possible dimer combinations (7). A large number of these pairs, but not all, are biochemically compatible (Ref. 12 and references therein). One possible consequence of the model in Fig. 11 would be that the Gbeta protein on CCT/TRiC provides an uncommitted reservoir of protein capable of responding to changing cellular requirements for different Gbeta{gamma} dimers, dependent upon production of the small and highly variable G{gamma} subunit. Thus, this mechanism of Gbeta{gamma} dimer production (Fig. 11) may provide a "proofreading" step whereby Gbeta remains in reserve on CCT/TRiC until a correct G{gamma} subunit is provided to stabilize its release from the complex. Through this, the folding of Gbeta by CCT/TRiC allows an additional level of regulation of the heterotrimeric G proteins based upon their inherent specificity for dimer assembly.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant DK37219 (to J. D. H.), the Medical Scientist Training Program of the Medical University of South Carolina, and a PhRMA Foundation fellowship award (to C. A. W.). This work was also supported by institutional support of the Medical University of South Carolina DNA Sequencing Facility and the Medical University of South Carolina Mass Spectrometry Facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, 303 BSB, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-3209; Fax: 843-792-3209; E-mail: hildebjd{at}musc.edu.

2 The abbreviations used are: TBS, Tris-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry; MS, mass spectrometric; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); ADPbetaS, adenosine 5'-O-2-(thiodiphosphate); PhLP, phosducin-like protein. Back

3 C. A. Wells, J. Dingus, and J. D. Hildebrandt, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Kevin Schey, Jennifer Bethard, John Cleator, Kathryn Robinson, Bronwyn Tatum, Lia Campbell, and Brook White for assistance and helpful discussions in the completion of this study. We also thank Drs. Henry Fong, Mel Simon, N. Gautam, William Simonds, and Nicholas Ryba for providing constructs and cDNAs. We also thank the Guthrie cDNA Resource Center (now the University of Missouri-Rolla cDNA Resource Center) for making available a number of the cDNA clones used in this study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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