title: Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner.
abstract: The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-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 Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 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.
authors.affiliation: Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Chih-Tien
authors.initials: CT
authors.lastname: Wang
authors.forename: Jihong
authors.initials: J
authors.lastname: Bai
authors.forename: Meyer B
authors.initials: MB
authors.lastname: Jackson
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002135:Calcium-Binding Proteins; D005089:Exocytosis / Q000235:genetics*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D055672:Static Electricity; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium