Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170.
GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.
Ohta
Eiji
E
Department of Cell Biology, Fukuoka University School of Medicine, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
Misumi
Yoshio
Y
Sohda
Miwa
M
Fujiwara
Toshiyuki
T
Yano
Akiko
A
Ikehara
Yukio
Y
D000215:Acylation; D001324:Autoantigens; D001483:Base Sequence; D006056:Golgi Apparatus / Q000737:chemistry* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000032:analysis; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques
D001324:Autoantigens; C108388:GOLGA3 protein, human; C480862:GOLGA7 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger