title: Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes.
abstract: Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.
authors.affiliation: Department of Medicine, Massachusetts General Hospital, Charlestown, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Brand
authors.forename: E J
authors.initials: EJ
authors.lastname: Holtzman
authors.forename: D A
authors.initials: DA
authors.lastname: Scher
authors.forename: D A
authors.initials: DA
authors.lastname: Ausiello
authors.forename: J L
authors.initials: JL
authors.lastname: Stow
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D018374:LLC-PK1 Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D037342:Pertussis Toxin; D011509:Proteoglycans / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000939:Epitopes; D009227:Myristic Acids; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D010455:Peptides; D011509:Proteoglycans; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; C085706:FLAG peptide; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins