Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIα), the original Syt VII, and two minor isoforms (Syt VIIβ and Syt VIIγ), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIα. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIα (65kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII—green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.
- C2 domain
- C-type tandem C2 protein
- dense-core vesicle
- membrane traffic
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AB075900-1.
Abbreviations used: CAP, adenylate cyclase-associated protein; C-type, C-terminal-type; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; LC—MS/MS, liquid chromatography-tandem mass spectrometry; NGF, nerve growth factor; RT, reverse transcriptase; Syt(s), synaptotagmin(s); TGN, trans-Golgi network.
- The Biochemical Society, London ©2002