Studies on Plasma Membrane Proteins Involved in Membrane Traffic: Syntaxins and E-Syts
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Fusion of synaptic vesicles is catalyzed by SNARE complex assembly which requires that the SNARE proteins syntaxin-1A and -1B, two isoforms of syntaxin-1, switch from a 'closed' to an 'open' conformation. To test the physiological significance of this switch, I analyzed mutant mice with a point mutation in syntaxin-1B which renders it predominantly 'open' in the syntaxin-1A null background. Whereas deletion of syntaxin-1A caused no detectable phenotype, opening of syntaxin-1B produced lethal epilepsy, independent of the presence of syntaxin-1A. Morphological and electrophysiological analyses revealed that opening of syntaxin-1B impaired steps in exocytosis upstream of vesicle priming, but enhanced Ca2+</sup -triggering of fusion-pore opening downstream of priming, indicating that the conformational switch from closed to open syntaxin-1 controls the entry of vesicles into, and their exit from the exocytosis reaction, leading to a model whereby vesicle priming is initiated by the closed conformation of syntaxin-1, but executed by opening of syntaxin-1. To understand the differential roles of syntaxin-1A and -1B, GFP-KI mice were generated, which unexpectedly phenocopied KO mice, suggesting functional inactivation by GFF-fusion. Interestingly, GFP-syntaxin-1A and -1B fusion proteins showed differential binding properties to its major binding partners, Munc18-1 and other SNARE proteins. While loss of syntaxin-1A resulted in no detectable phenotype, syntaxin-1B GFP-KI mice died at P14 and showed major phenotype, such as unbalanced coordination of their body with shortening of cerebellar purkinje cell layer and specific innervations on neuromuscular junction synapse, suggesting possible mechanisms for lethality caused by loss of syntain-1B. C2-domains are autonomously folded protein modules that generally function as Ca2+- and phospholipid-binding domains, usually involved in membrane trafficking. A family of evolutionarily conserved mammalian proteins, referred to as E-Syts (Extended Synaptotagmin-like proteins), contains an N-terminal transmembrane region, and five (ESyt1) or three (E-Syt2 and E-Syt3) C-terminal C2-domains. In vitro phospholipid binding assay showed the first C2-domain of E-Syt2 was capable of Ca2+ -dependent phospholipid binding, suggesting that E-Syts bind Ca2+ via their first C2-domain in a phospholipid complex. Expression of myc-tagged E-Syt proteins demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3 in a C2C-domaindependent manner, revealing an unexpected mechanism by C2C-domain functions as a targeting motif, independent of transmembrane region.