Abstract
Gβγ dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, 35S-labeled Gγ subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, 35S-labeled Gβ subunits traveled at a high apparent molecular mass (∼700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gβ isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gβ subunits to efficiently form dimers with Gγ. When translated Gγ was added to translated Gβ, a new band of low apparent molecular mass (∼50 kDa) was observed, which was labeled by either 35S-labeled Gβ or Gγ, indicating that it is a dimer. Formation of the Gβγ 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 Gβ with CCT/TRiC. Although Gγ did not bind CCT/TRiC, addition of Gγ to previously synthesized Gβ caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gβ subunits and that CCT/TRiC mediates Gβγ dimer formation by an ATP-dependent reaction.
Highlights
Isolated CCT/TRiC subunits were identified as interacting partners of the yeast G 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
No G5 was detectable in immunoprecipitates of FLAG-G␥3 coincubated with this G, indicating that G5 forms dimers very poorly with G␥3
In immunoprecipitates with G3, much less G3 was present than was seen with G1, G2, or G4, but G3 was clearly present as compared with G5 or the control lanes
Summary
Vectors—All G and FLAG-G␥ constructs were as described previously [12]. All FLAG-G constructs were obtained from the Guthrie cDNA Resource Center (available at www.cdna.org). The beads were blocked against nonspecific binding by incubation for 1 h at room temperature in 10 volumes of TBSBC with 5% reticulocyte lysate and washed twice with 50 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% C12E10 (TBSC). 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␣ antibodies diluted in Odyssey blocking buffer (1:1 with TBST containing 0.2% Tween 20). Proteomics Analysis of Samples Recovered after SDS-PAGE— FLAG-tagged G1 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 -mercaptoethanol and was run on 4 –20% Criterion gels. For the rabbit data base, this included all identified peaks with protein scores Ͼ44, whereas for the rodent data base, this included peaks with protein scores Ͼ63
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