Identification and characterization of GIV, a novel Galpha i/s-interacting protein found on COPI, endoplasmic...

In this report, we characterize GIV (Galpha-interacting vesicle-associated protein), a novel protein that binds members of the Galpha(i) and Galpha subfamilies of heterotrimeric G proteins. The Galpha(s) interaction site was mapped to an 83-amino acid region of GIV that is enriched in highly charged amino acids. BLAST searches revealed two additional mammalian family members, Daple and an uncharacterized protein, FLJ00354. These family members share the highest homology at the Galpha binding domain, are homologous at the N terminus and central coiled coil domain but diverge at the C terminus. Using affinity-purified IgG made against two different regions of the protein, we localized GIV to COPI, endoplasmic reticulum (ER)-Golgi transport vesicles concentrated in the Golgi region in GH3 pituitary cells and COS7 cells. Identification as COPI vesicles was based on colocalization with beta-COP, a marker for these vesicles. GIV also codistributes in the Golgi region with endogenous calnuc and the KDEL receptor, which are cis Golgi markers and with Galpha(i3)-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with beta-COP and Galpha(i3) on vesicles found in close proximity to ER exit sites and to cis Golgi cisternae. In cell fractions prepared from rat liver, GIV is concentrated in a carrier vesicle fraction (CV2) enriched in ER-Golgi transport vesicles. beta-COP and several Galpha subunits (Galpha(i1-3), Galpha(s)) are also most enriched in CV2. Our results demonstrate the existence of a novel Galpha-interacting protein associated with COPI transport vesicles that may play a role in Galpha-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.