Identification and Characterization of GCP16, a Novel Acylated Golgi Protein That Interacts with GCP170

Abstract

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 NH 2 -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. 69 and Cys 72 , 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. Labeling experiments with [ 3 H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys

DHHC9 and GCP16 Constitute a Human Protein Fatty Acyltransferase with Specificity for H- and N-Ras

“…GCP16 is a 137-amino acid protein with no obvious structural features other than a short predicted coiled-coil domain. Ohta et al (25) found that GCP16 is palmitoylated and that fatty acylation accounts for the behavior of GCP16 as an integral membrane protein and its association with the Golgi complex. Overexpression of GCP16 in COS cells impaired trafficking of vesicular stomatitis virus glycoprotein to the cell surface and modestly inhibited secretion of a soluble form of dipeptidyl peptidase IV without having an obvious effect on Golgi morphology (25).…”

“…GCP16 was previously identified as a palmitoylated protein that interacts with the Golgi autoantigen GCP170 (25). GCP170 is a member of the golgin family, high molecular weight proteins with cytoplasmic coiled-coil domains that are localized to the cytoplasmic face of the Golgi cisternae (40).…”

“…The DHHC9 transcript was absent from thymus, spleen, and peripheral blood leukocytes. A previous study reported that GCP16 mRNA is also expressed ubiquitously (25) (Fig. 2B).…”

“…1C. The mammalian candidate for an Erf4 homolog is identical to GCP16 (Golgi-complex associated protein of 16 kDa), a palmitoylated protein that interacts with the Golgiassociated protein GCP170 (25). We obtained full-length cDNAs for DHHC9 and GCP16 to investigate whether they encoded functional orthologs of Erf2 and Erf4.…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Recent elegant fluorescence experiments revealed that depalmitoylated H-and N-Ras rapidly traffic from the cell surface to the Golgi via a non-vesicular pathway [64,65]. The palmitoyltransferase complex responsible for palmitoylating Ras was recent identified as DHHC9/GCP16 (Erf2/Erf4 in yeast) and these have also been localised to the Golgi [66][67][68]. Taken together these data suggest a model where palmitoylation in the Golgi stabilises Ras-membrane interactions facilitating trafficking to the plasma membrane before depalmitoylation causes Ras to become cytosolic and traffic back to the ER/Golgi.…”