Abstract
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.
Highlights
Over 60 different Rab proteins are described in mammalian cells as key modulators of transport vesicles in the exocytotic and endocytotic pathway [1]
The subfamily of Rab3 proteins consists of four isoforms that are all expressed in secretory cells [2] and that bind to synaptic and other secretory vesicles in the active, GTP-bound state [3,4]
GTP-Rab3 isoforms interact with several proteins involved in vesicle secretion like Rabphilin3a and RIM [5,6] and dissociate from these proteins and the vesicle upon GTP hydrolysis [7,8]
Summary
Over 60 different Rab proteins are described in mammalian cells as key modulators of transport vesicles in the exocytotic and endocytotic pathway [1]. The subfamily of Rab3 proteins consists of four isoforms that are all expressed in secretory cells [2] and that bind to synaptic and other secretory vesicles in the active, GTP-bound state [3,4]. In yeast and in endosome fusion, Rab proteins regulate vesicle docking in conjunction with Sec1/Munc18 proteins [15,16] and in PC12 cells, Rab3a overexpression increases docking while knockdown leads to fewer vesicles near the membrane [17,18].
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