Phosphorylation of the Vesicle Docking Protein p115 Regulates Its Association with the Golgi Membrane

Abstract

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...

Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis

“…Phosphorylation of p115 enhances this binding (6), whereas phosphorylation of GM130 inhibits it (7). During mitosis, p115 is dephosphorylated by an unknown phosphatase (8), whereas GM130 is phosphorylated by the mitotic kinase Cdc2 (9). This suggests that the vesiculation of the Golgi apparatus that occurs during cell division may be caused by inhibition of p115 tethering such that Golgi vesicles form but do not dock and fuse (7).…”

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages

“…33 Phosphorylated USO1 is found in the cytosol, whereas the unphosphorylated molecule locates on membranes of the Golgi complex and transcytotic vesicles. 34 This study demonstrated interaction between MIF and USO1, as well as colocalization within vesicles of human peripheral blood mononuclear cells (PBMC). Moreover, USO1 was required for the secretion of MIF in response to LPS and was released alongside MIF.…”

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

“…Construction and Transfection of Expression Plasmids-cDNAs encoding the entire coding region of GCP60 (wild type) and B8 were ligated to a position downstream of the sequence encoding the Met-FLAG tag in pSG5 expression vector so that the products could be recognized by the monoclonal anti-FLAG antibody M2 (28,31). The COOH-terminally deleted mutant M1 was prepared by introducing a termination codon into an appropriate site of the wild type plasmid.…”

“…The mutant cDNA sequences (D1ϪD3) obtained were verified and ligated in frame to pEGFP-1 for GFP-fused proteins. The plasmid for N-acetylglucosaminyltransferase fused with GFP (NAGFP) (32) (17,31). Cell lysates were prepared and subjected to immunoprecipitation with polyclonal anti-GCP60 as described previously (17,31).…”