The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity.
ABSTRACT The vesicle-tethering protein p115 functions in endoplasmic reticulum-Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini-stacked Golgi fragments, and gathered around microtubule-organizing center in a microtubule-dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis-Golgi vesicle-tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re-expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis-Golgi in vesicular trafficking.
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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.Journal of Biological Chemistry 12/2001; 276(48):45298-306. · 4.65 Impact Factor
Article: Golgi architecture and inheritance.[show abstract] [hide abstract]
ABSTRACT: Golgi inheritance proceeds via sequential biogenesis and partitioning phases. Although little is known about Golgi growth and replication (biogenesis), ultrastructural and fluorescence analyses have provided a detailed, though still controversial, perspective of Golgi partitioning during mitosis in mammalian cells. Partitioning requires the fragmentation of the juxtanuclear ribbon of interconnected Golgi stacks into a multitude of tubulovesicular clusters. This process is choreographed by a cohort of mitotic kinases and an inhibition of heterotypic and homotypic Golgi membrane-fusion events. Our model posits that accurate partitioning occurs early in mitosis by the equilibration of Golgi components on either side of the metaphase plate. Disseminated Golgi components then coalesce to regenerate Golgi stacks during telophase. Semi-intact cell and cell-free assays have accurately recreated these processes and allowed their molecular dissection. This review attempts to integrate recent findings to depict a more coherent, synthetic molecular picture of mitotic Golgi fragmentation and reassembly. Of particular importance is the emerging concept of a highly regulated and dynamic Golgi structural matrix or template that interfaces with cargo receptors, Golgi enzymes, Rab-GTPases, and SNAREs to tightly couple biosynthetic transport to Golgi architecture. This structural framework may be instructive for Golgi biogenesis and may encode sufficient information to ensure accurate Golgi inheritance, thereby helping to resolve some of the current discrepancies between different workers.Annual Review of Cell and Developmental Biology 02/2002; 18:379-420. · 17.98 Impact Factor
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ABSTRACT: We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.Journal of Biological Chemistry 09/1986; 261(24):11398-403. · 4.65 Impact Factor