A role for GRIP domain proteins and/or their ligands in structure and function of the trans Golgi network

Abstract

TGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. We have probed for GRIP protein function by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy we found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization.