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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 -Dimer Assembly^*

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

G dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, ³⁵S-labeled G subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, ³⁵S-labeled G subunits traveled at a high apparent molecular mass (700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-G isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different G subunits to efficiently form dimers with G. When translated G was added to translated G, a new band of low apparent molecular mass (50 kDa) was observed, which was labeled by either ³⁵S-labeled G or G, indicating that it is a dimer. Formation of the G 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 G with CCT/TRiC. Although G did not bind CCT/TRiC, addition of G to previously synthesized G caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds G subunits and that CCT/TRiC mediates G dimer formation by an ATP-dependent reaction.

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

^* 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.

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.

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