Identification and Characterization of a Novel Golgi Protein, GCP60, That Interacts with the Integral Membrane Protein Giantin

Abstract

We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.

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

“…Data base searches show that GCP16 has 96.6% identity in amino acid sequence to an unannotated gene product of mouse (GenBank TM accession number AB041568.1) and a significant similarity to those of D. melanogaster (accession number AE003756.10) and C. elegans (accession number Z99281.1) with identities of 53.7 and 32.8%, respectively. No homologous sequence, however, is found in yeast and plants, indicating that GCP16 is absent in yeast and plants, as reported for GCP60 and giantin (35). This is unusual because most vesicular transport factors and Golgi components are evolutionarily conserved (43).…”

“…In contrast, the overexpression of the wild type GCP16 caused the inhibitory effect on the transport of VSV-G protein from the Golgi to the cell surface. This is considered to be a dominant-negative effect of the overexpression of GCP16, as observed in other proteins including GCP60 (35).…”

“…COS-1 cells were transfected with the pSG5 plasmid containing the cDNA construct of VSV-G/ts045 fused to green fluorescent protein (GFP-VSV-G/ts045) (35). After incubation at 39.5°C for 20 h, the cells were transferred to the permissive temperature at 32°C and incubated for an additional 2 h. The cells were fixed and observed for the GFP image.…”

“…Immunocytochemistry-HeLa or COS-1 cells with or without transfection were subjected to immunofluorescence microscopy as described previously (30,35). When indicated, the cells were treated with brefeldin A (2 g/ml) at 37°C before fixation and immunostaining.…”

N4WBP5, a Potential Target for Ubiquitination by the Nedd4 Family of Proteins, Is a Novel Golgi-associated Protein

“…These results indicate that the ectopic expression of N4WBP5 causes a disruption of the Golgi structure and that the altered localization of N4WBP5 and GM130 in N4WBP5-transfected cells is probably to be attributed to the disruption of the Golgi complex. As overexpression of several Golgi-associated proteins, such as giantin and GCP60, has been shown to disrupt Golgi structure (35)(36)(37), our results suggest that N4WBP5 is involved in the structural maintenance of the Golgi complex. Consistent with our prediction noted above, no significant nuclear localization of N4WBP5 was apparent in transfected cells.…”