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.

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 microtubuledependent 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.

ABSTRACT. When increased production of secretory proteins overwhelms the capacity of the endoplasmic reticulum (ER) and the Golgi apparatus, eukaryotic cells expand their capacity to sustain secretory function. The capacity of the ER is enhanced by the mechanism called the ER stress response, but the mechanism regulating Golgi capacity (the Golgi stress response) has remained unclear. Here, we found that transcription of Golgirelated genes, including glycosylation enzymes as well as factors involved in post-Golgi vesicular transport and maintenance of Golgi structure, was upregulated upon treatment with monensin, an ionophore that disrupts the function of acidic organelles, including the Golgi apparatus and lysosomes by neutralizing their lumen. This transcriptional induction was found to be commonly regulated by a novel cis-acting element called the Golgi apparatus stress response element (GASE), whose consensus sequence is ACGTGgc. When the function of the Golgi apparatus was specifically disturbed by overexpression of GCP60, a Golgi-localized protein that binds to giantin, transcription from GASE was significantly induced. These results suggest that mammalian cells have the Golgi stress response, and that GASE regulates transcriptional induction involved in the Golgi stress response.

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The coiled-coil Golgi membrane protein golgin-84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin-84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin-84 as the tether for COPI vesicles of intra-Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin-84 knockdown (KD) cells. The depletion of golgin-84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and cis-Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex-dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin-84. Surprisingly, the interaction between golgin-84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin-84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin-84 with COG plays an important role in the tethering process of intra-Golgi retrograde vesicle traffic.

The vesicle docking protein p115 was found to be phosphorylated in a cell cycle-specific manner; it was found phosphorylated in interphase but not in mitotic cells. During interphase, however, two forms of p115 were detected in the cells; the phosphorylated form was found exclusively in cytosol, whereas the unphosphorylated form was associated with membranes, mostly of the Golgi complex. The latter form was released from the membranes upon phosphorylation. Mutational analysis revealed that the phosphorylation site of p115 was the Ser 942 residue in the C-terminal acidic domain. A mutant with a single substitution of Ser 942 3 Ala markedly increased its association with the Golgi membrane. Another mutant with Ser 942 3 Asp was able to associate with the membrane, although at a decreased level, indicating that the dissociation of p115 from the membrane is not simply due to the negative charge of phosphorylated Ser 942 . Taken together, these results suggest that the phosphorylation of Ser 942 at the C-terminal acidic domain regulates the interaction of p115 with the Golgi membrane, possibly taking part in the regulatory mechanism of vesicular transport.Vesicular transport of proteins is carried out by the formation of coated vesicles from a donor compartment, followed by their uncoating and subsequent docking and fusion of the vesicles with a target compartment membrane, in which a number of soluble and membrane proteins are involved (1). The docking of vesicles to target membranes is accomplished through the specific interaction between membrane proteins named v-and t-SNAREs (vesicle and target SNAP receptors) (1, 2). This is followed by binding of SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins) and N-ethylmaleimide-sensitive factor, which lead to membrane fusion. Vesicle docking is also controlled by interactions of SNAREs with other proteins including Rab proteins (3).p115, a peripheral membrane protein localized to the Golgi apparatus and also present in the cytoplasm, was first identified as a component required for intra-Golgi transport (4) and found to be identical to the transcytosis-associated protein TAP (5). Structural analysis also indicates that it is a homolog to Uso1p, a yeast protein required for transport from the endoplasmic reticulum to the Golgi (6). p115/TAP exits as a parallel homodimer with two globular heads followed by a rod-like domain containing a C-terminal acidic tail (5, 7). p115/TAP and Uso1p have been shown to function in a docking step prior to membrane fusion (8). Recently, Warren and his colleagues (9) demonstrated that p115 binds to Golgi membranes with high affinity during interphase but not during mitosis. In addition, GM130, a peripheral protein tightly associated with the cisGolgi membrane (10), was identified as the binding site for p115 on the Golgi membrane (11). GM130 was modified by phosphorylation only during mitosis, resulting in no binding of GM130 to p115.The mechanism of membrane binding inhibition by p115 during mitosis would provide a molecu...

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 NH 2 -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 ␣-helical domain that likely forms a coiledcoil between non-coil domains at the NH 2 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 nonclathrin-coated vesicles (␤-COP, a subunit of coatomer), but did not co-localize with ␤-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.

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