Wang, PingPingWangCHIH-TIEN WANGBai, JihongJihongBaiJackson, Meyer B.Meyer B.JacksonChapman, Edwin R.Edwin R.Chapman2019-07-022019-07-022003-11-2100219258https://scholars.lib.ntu.edu.tw/handle/123456789/412529The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca 2+ -triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca 2+ . Upon binding Ca 2+ , positively charged residues within the Ca 2+ -binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca 2+ requirements for syt-liposome interactions from ∼20 to ∼40 μM Ca 2+ . Kinetic analysis revealed that the reduction in Ca 2+ -sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in a ∼50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.enjournal article10.1074/jbc.M306728200129637432-s2.0-0344012489WOS:000186569400099https://api.elsevier.com/content/abstract/scopus_id/0344012489