Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.
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 VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. 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. 65 kDa 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 VIIalpha (65 kDa) 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.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Ogata
Yukie
Y
Saegusa
Chika
C
Kanno
Eiko
E
Mikoshiba
Katsuhiko
K
D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D016716:PC12 Cells; D020033:Protein Isoforms / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism; D050857:Synaptotagmins; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C484231:CAP2 protein, human; D002135:Calcium-Binding Proteins; C081782:Cap1 protein, rat; C484230:Cap2 protein, rat; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C494302:SYT7 protein, human; C494303:Syt7 protein, mouse; C494304:Syt7 protein, rat; D050857:Synaptotagmins; D004247:DNA