Discrete residues in the c(2)b domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size.
It has been demonstrated that synapses lacking functional synaptotagmin I (Syt I) have a decreased rate of synaptic vesicle endocytosis. Beyond this, the function of Syt I during endocytosis remains undefined. Here, we demonstrate that a decreased rate of endocytosis in syt(null) mutants correlates with a stimulus-dependent perturbation of membrane internalization, assayed ultrastructurally. We then separate the mechanisms that control endocytic rate and vesicle size by mapping these processes to discrete residues in the Syt I C(2)B domain. Mutation of a poly-lysine motif alters vesicle size but not endocytic rate, whereas the mutation of calcium-coordinating aspartate residues (syt-D3,4N) alters endocytic rate but not vesicle size. Finally, slowed endocytic rate in the syt-D3,4N animals, but not syt(null) animals, can be rescued by elevating extracellular calcium concentration, supporting the conclusion that calcium coordination within the C(2)B domain contributes to the control of endocytic rate.
Poskanzer
Kira E
KE
Department of Biochemistry and Biophysics, University of California, San Francisco, Rock Hall, 1550 Fourth Street, Box 2822, San Francisco, California 94158, USA.
Fetter
Richard D
RD
Davis
Graeme W
GW
D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D004558:Electric Stimulation / Q000379:methods; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D049452:Green Fluorescent Proteins / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D007814:Larva; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009154:Mutation; D010775:Photic Stimulation / Q000379:methods; D017434:Protein Structure, Tertiary; D013569:Synapses / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000502:physiology* / Q000648:ultrastructure; D050863:Synaptotagmin I / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D033965:Adaptor Protein Complex alpha Subunits; D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; D050863:Synaptotagmin I; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D008239:Lysine; D002118:Calcium