Identification and characterization of GIV, a novel Gαi/s- interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles

Helen Le-Niculescu, Ingrid Niesman, Thierry Fischer, Luc DeVries, Marilyn G. Farquhar

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Abstract

In this report, we characterize GIV (Gα-interacting vesicle-associated protein), a novel protein that binds members of the Gαi and Gαs subfamilies of heterotrimeric G proteins. The Gα 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 Gα 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 β-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 Gαi3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with β-COP and Gαi3 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. β-COP and several Gα subunits (Gαi1-3, Gαs) are also most enriched in CV2. Our results demonstrate the existence of a novel Gα-interacting protein associated with COPI transport vesicles that may play a role in Gα-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.

Original languageEnglish (US)
Pages (from-to)22012-22020
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number23
DOIs
StatePublished - Jun 10 2005
Externally publishedYes

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Coat Protein Complex I
Transport Vesicles
Endoplasmic Reticulum
Proteins
Amino Acids
Heterotrimeric GTP-Binding Proteins
Immunoelectron Microscopy
Liver
Rats

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification and characterization of GIV, a novel Gαi/s- interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles. / Le-Niculescu, Helen; Niesman, Ingrid; Fischer, Thierry; DeVries, Luc; Farquhar, Marilyn G.

In: Journal of Biological Chemistry, Vol. 280, No. 23, 10.06.2005, p. 22012-22020.

Research output: Contribution to journalArticle

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abstract = "In this report, we characterize GIV (Gα-interacting vesicle-associated protein), a novel protein that binds members of the Gαi and Gαs subfamilies of heterotrimeric G proteins. The Gα 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 Gα 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 β-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 Gαi3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with β-COP and Gαi3 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. β-COP and several Gα subunits (Gαi1-3, Gαs) are also most enriched in CV2. Our results demonstrate the existence of a novel Gα-interacting protein associated with COPI transport vesicles that may play a role in Gα-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.",
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