The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion

Max Planck Institute for Experimental Medicine, Gottingen, Germany.
Nature (Impact Factor: 42.35). 07/1997; 387(6635):810-4. DOI: 10.1038/42954
Source: PubMed

ABSTRACT The Rab family of low-molecular-mass GTP-binding proteins are thought to guide membrane fusion between a transport vesicle and the target membrane, and to determine the specificity of docking. The docking and fusion of vesicles is, however, a complex multistep reaction, and the precise point at which Rab proteins act in these sequential processes is unknown. In brain, the Rab protein Rab3A is specific to synaptic vesicles, whose exocytosis can be monitored with submillisecond resolution by following synaptic transmission. We have now determined the precise point at which Rab3A acts in the sequence of synaptic vesicle docking and fusion by using electrophysiological analysis of neurotransmitter release in Rab3A-deficient mice. Unexpectedly, the size of the readily releasable pool of vesicles is normal, whereas Ca2+-triggered fusion is altered in the absence of Rab3A in that a more-than-usual number of exocytic events occur within a brief time after arrival of the nerve impulse.

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    • "Thus, despite a wealth of genetic, overexpression, electrophysiological, and biochemical data regarding Rab3s (A, B, C, and D), it is difficult to formulate a coherent hypothesis about the functions of these proteins in membrane fusion. Rab3A has been claimed to maintain the supply of vesicles into the releasable pool (Coleman and Bykhovskaia, 2009) and to be unnecessary for refilling this pool (Geppert et al., 1997; Leenders et al., 2001); to increase the docking of vesicles to the plasma membrane (Tsuboi and Fukuda, 2006; Coleman et al., 2007; van Weering et al., 2007) and not to be involved in docking but in the recruitment of vesicles (Leenders et al., 2001; Tian et al., 2012); to regulate the number of vesicles that fuse during stimulation (Wang et al., 2008) and the amount of transmitter in a quantum (Geppert et al., 1997; Coleman and Bykhovskaia, 2009); to be unnecessary for fusion itself (Johannes et al., 1994) but to control exocytosis of vesicles through direct interaction with fusion pores of unusual characteristics (Wang et al., 2008); to limit evoked release to a single quantum for each release site during a single impulse (Johannes et al., 1998); to participate in priming (Dulubova et al., 2005; Huang et al., 2011), etc. Perhaps the only notion all studies agree on is that Rab3A modulates exocytosis. "
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    ABSTRACT: Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
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    • "The levels of many other synaptic proteins, including GDI – known to bind RAB3A – were found not altered. In a later work, (Geppert et al., 1997) further characterized Rab3a-null mice and reported that the readily releasable pool of vesicles is normal in Rab3a-null mice, whereas the Ca 2+ triggered fusion is altered in the absence of RAB3A. In another work it was shown that in hippocampal CA3 mossy fiber synapses LTP and LTD were abolished in Rab3a-null mutant mice (Castillo et al., 1997). "
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    • "RAB3A is an abundant synaptic vesicle protein that regulates neurotransmission (Sudhof, 2004). Mouse Rab3a inactivation results in increased hippocampal paired-pulse facilitation (Geppert et al., 1997), while RAB3A overexpression in cell culture activates spontaneous exocytosis (Schluter et al., 2002), similar to PC12 cells transfected with expanded DMPK constructs. Given the involvement of RAB3A in short-term synaptic plasticity and neurotransmitter release , RAB3A upregulation in DMSXL mice likely contributes to altered paired-pulse facilitation and mediates the increase in basal neurosecretion in transfected PC12 cells. "
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