Identification and characterization of a novel C2B splice variant of synaptotagmin I.
We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-beta, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-alpha. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-beta exon in the nervous system. Synaptotagmin I C2B-beta showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-alpha, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-beta with a higher affinity than synaptotagmin I C2B-alpha. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.
Nakhost
Arash
A
Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, Montreal, QC, Canada.
Houeland
Gry
G
Blandford
Vanessa E
VE
Castellucci
Vincent F
VF
Sossin
Wayne S
WS
D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D002918:Chymotrypsin / Q000737:chemistry; D015951:Factor Xa / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002918:Chymotrypsin; D015951:Factor Xa; D002118:Calcium