title: Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN.
abstract: The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network (TGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin-245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.
authors.affiliation: The Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Merran C
authors.initials: MC
authors.lastname: Derby
authors.forename: Catherine
authors.initials: C
authors.lastname: van Vliet
authors.forename: Darren
authors.initials: D
authors.lastname: Brown
authors.forename: Michael R
authors.initials: MR
authors.lastname: Luke
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
authors.forename: Wanjin
authors.initials: W
authors.lastname: Hong
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stow
authors.forename: Paul A
authors.initials: PA
authors.lastname: Gleeson
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D001324:Autoantigens / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology; D026901:Membrane Transport Proteins / Q000502:physiology*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012799:Sialyltransferases / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D021601:trans-Golgi Network / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D001324:Autoantigens; C472298:GCC1 protein, human; C472299:GCC2 protein, human; C098772:GOLGA4 protein, human; D000077614:Golgi Matrix Proteins; C450721:Golgi complex autoantigen, 97-kDa; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D012799:Sialyltransferases; C097313:ADP-ribosylation factor related proteins; D020727:ADP-Ribosylation Factors; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase