Identification and characterization of GIV, a novel Galpha i/s-interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles.
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.
Le-Niculescu
Helen
H
Department of Cellular and Molecular Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Niesman
Ingrid
I
Fischer
Thierry
T
DeVries
Luc
L
Farquhar
Marilyn G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D015152:Blotting, Northern; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D020756:Coatomer Protein / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D019992:Databases as Topic; D016208:Databases, Factual; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000502:physiology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016716:PC12 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D014162:Transfection; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000096:biosynthesis / Q000502:physiology*
D001426:Bacterial Proteins; C502451:CCDC88A protein, human; D020756:Coatomer Protein; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D033921:Vesicular Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D005982:Glutathione Transferase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs