A PUTATIVE HETEROTRIMERIC G-PROTEIN INHIBITS THE FUSION OF COPI-COATED VESICLES - SEGREGATION OF HETEROTRIMERIC G-PROTEINS FROM COPI-COATEDVESICLES

Heterotrimeric G proteins have been implicated in the regulation of in tracellular protein transport, but their mechanism of action remains u nclear, bn vivo, secretion of chromogranin B, tagged with the green fl uorescent protein, was inhibited by the addition of a general activato r of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Gels apparatu s. In an in vitro assay that reconstitutes intra-Golgi protein transpo rt, we find that a membrane-bound and AlF4--sensitive factor is involv ed in the fusion reaction. To determine whether this effect is mediate d by a heterotrimeric G protein localized to COPI-coated transport ves icles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Becau se G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found th at trimeric G proteins from isolated Golgi membranes were partially in soluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Gels-derived detergent-insol uble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.