Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans
| Autor: | Janet S. Duerr, Gregory P. Mullen, James B. Rand, John McManus, Eleanor A. Mathews, Angie Duke, Jennifer Gaskin, John A. Crowell |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Central Nervous System
Gene isoform Recombinant Fusion Proteins Molecular Sequence Data Fluorescent Antibody Technique Polymerase Chain Reaction Article Synaptotagmin 1 Animals Genetically Modified Cellular and Molecular Neuroscience Image Processing Computer-Assisted Animals Protein Isoforms Amino Acid Sequence Caenorhabditis elegans Caenorhabditis elegans Proteins Promoter Regions Genetic Molecular Biology Gene Peptide sequence Alleles C2 domain Genetics Microscopy Confocal biology Alternative splicing Cell Biology biology.organism_classification Fusion protein Alternative Splicing Protein Transport Synaptotagmin I Mutagenesis Site-Directed |
| Zdroj: | Molecular and Cellular Neuroscience. 34:642-652 |
| ISSN: | 1044-7431 |
| DOI: | 10.1016/j.mcn.2007.01.009 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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