Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans.
Synaptotagmin 1, encoded by the snt-1 gene in Caenorhabditis elegans, is a major synaptic vesicle protein containing two Ca(2+)-binding (C2) domains. Alternative splicing gives rise to two synaptotagmin 1 isoforms, designated SNT-1A and SNT-1B, which differ in amino acid sequence in the third, fourth, and fifth beta-strands of the second C2 domain (C2B). We report here that expression of either SNT-1 isoform under control of a strong pan-neural promoter fully rescues the snt-1 null phenotype. Furthermore, C-terminal fusions of either isoform with GFP are trafficked properly to synapses and are fully functional, unlike synaptotagmin 1Colon, two colonsGFP fusions in mice. Analysis of isoform expression with genomic GFP reporter constructs revealed that the SNT-1A and-1B isoforms are differentially expressed and localized in the C. elegans nervous system. We also report molecular, behavioral, and immunocytochemical analyses of twenty snt-1 mutations. One of these mutations, md259, specifically disrupts expression of the SNT-1A isoform and has defects in a subset of synaptotagmin 1-mediated behaviors. A second mutation, md220, is an in-frame 9-bp deletion that removes a conserved tri-peptide sequence (VIL) in the second beta-strand of the C2B domain and disrupts the proper intracellular trafficking of synaptotagmin. Site-directed mutagenesis of a functional SNT-1Colon, two colonsGFP fusion protein was used to examine the potential role of the VIL sequence in synaptotagmin trafficking. Although our results suggest the VIL sequence is most likely not a specific targeting motif, the use of SNT-1Colon, two colonsGFP fusions has great potential for investigating synaptotagmin trafficking and localization.
Mathews
Eleanor A
EA
Program in Molecular, Cell and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
Mullen
Gregory P
GP
Crowell
John A
JA
Duerr
Janet S
JS
McManus
John R
JR
Duke
Angie
A
Gaskin
Jennifer
J
Rand
James B
JB
D000483:Alleles; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism*; D002490:Central Nervous System / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I / Q000235:genetics* / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I