The role of Rab3a in secretory vesicle docking requires association/dissociation of guanidine phosphates and Munc18-1.
Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1.
van Weering
Jan R T
JR
Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.
Toonen
Ruud F
RF
Verhage
Matthijs
M
D000818:Animals; D001921:Brain / Q000502:physiology*; D019439:Chromaffin Cells / Q000502:physiology*; D003001:Cloning, Molecular; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D019791:Guanidine / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D051938:Munc18 Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000502:physiology* / Q000648:ultrastructure; D012672:Semliki forest virus / Q000235:genetics; D020826:rab3A GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D051938:Munc18 Proteins; D049452:Green Fluorescent Proteins; D020826:rab3A GTP-Binding Protein; D019791:Guanidine