Molecular characterization of GCP170, a 170-kDa protein associated with the cytoplasmic face of the Golgi membrane.
We have isolated a cDNA clone encoding a protein (designated GCP170) of 1530 amino acid residues with a calculated molecular mass of 170 kDa that is localized to the Golgi complex. Hydropathy analysis shows that GCP170 contains no NH2-terminal signal sequence nor a hydrophobic domain sufficient for participating in membrane localization. It is also predicted that GCP170 has characteristic secondary structures including an extremely long alpha-helical domain that likely forms a coiled-coil between non-coil domains at the NH2 and COOH termini, suggesting that the protein is organized as a globular head, a stalk, and a tail. Immunocytochemical observations revealed that GCP170 was localized to the Golgi complex and the cytoplasm, consistent with biochemical data indicating that the protein exits as a membrane-associated form and a soluble form. GCP170 was dissociated from the Golgi membrane in response to brefeldin A as rapidly as a coat protein complex of non-clathrin-coated vesicles (beta-COP, a subunit of coatomer), but did not co-localize with beta-COP on the Golgi membrane when examined by immunoelectron microscopy. The protein was detected as phosphorylated and unphosphorylated forms, of which the unphosphorylated form was more tightly associated with the Golgi membrane. When cells were extracted with 1% Triton X-100 under microtubule-stabilizing conditions, GCP170 remained in the cells in association with the Golgi complex. These results indicate that GCP170 is a peripheral membrane protein with a long coiled-coil domain that may be involved in the structural organization or stabilization of the Golgi complex.
Misumi
Y
Y
Department of Biochemistry, Fukuoka University School of Medicine, Jonan-ku, Fukuoka 814-80, Japan.
Sohda
M
M
Yano
A
A
Fujiwara
T
T
Ikehara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D020126:Brefeldin A; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D001324:Autoantigens; D003517:Cyclopentanes; D018076:DNA, Complementary; C108388:GOLGA3 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D020126:Brefeldin A