Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation.
The role of heterotrimeric G-proteins on the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN) of PC12 cells was investigated. Using immunofluorescence and subcellular fractionation in conjunction with immunoblotting or ADP-ribosylation by either pertussis toxin or cholera toxin, TGN membranes were found to contain not only several alpha i/alpha o G-protein subunits including apparently alpha i3, but also alpha s. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alpha i/alpha o, a modification known to prevent their coupling to receptors, led to the stimulation of cell-free CSV and ISG formation, suggesting the presence of a guanine nucleotide exchange factor for alpha i/alpha o on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alpha i/alpha o, inhibited cell-free vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alpha s by cholera toxin treatment of cells resulted in a stimulation of cell-free CSV and ISG formation. This stimulation could be reversed when the alpha subunits not activated by cholera toxin, i.e. alpha i/alpha o, were activated by GTP gamma S and [AIF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of secretory vesicle formation.
Leyte
A
A
Institute for Neurobiology, University of Heidelberg, Germany.
Barr
F A
FA
Kehlenbach
R H
RH
Huttner
W B
WB
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002474:Cell-Free System; D002772:Cholera Toxin / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D016716:PC12 Cells; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins