Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells.
Synaptotagmins (Syts) III, V, VI, and X are classified as a subclass of Syt, based on their sequence similarities and biochemical properties (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273; Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Although they have been suggested to be involved in vesicular trafficking, as in the role of the Syt I isoform in synaptic vesicle exocytosis, their exact functions remain to be clarified, and even their precise subcellular localization is still a matter of controversy. In this study, we established rat pheochromocytoma (PC12) cell lines that stably express Syts III-, V-, VI-, and X-GFP (green fluorescence protein) fusion proteins, respectively, to determine their precise subcellular localizations. Surprisingly, Syts III-, V-, VI-, and X-GFP proteins were found to be targeted to specific organelles: Syt III-GFP to near the plasma membrane, Syt V-GFP to dense-core vesicles, Syt VI-GFP to endoplasmic reticulum-like structures, and Syt X-GFP to vesicles (other than dense-core vesicles) present in cytoplasm. We showed that Syt V-containing vesicles at the neurites of PC12 cells were processed to exocytosis in a Ca2+-dependent manner. Immunohistochemical analysis further showed that endogenous Syt V was also localized on dense-core vesicles in the mouse brain and specifically expressed in glucagon-positive alpha-cells in mouse pancreatic islets, but not in beta- or delta-cells. Based on these results, we propose that Syt V is a dense-core vesicle-specific Syt isoform that controls a specific type of Ca2+-regulated secretion.
Saegusa
Chika
C
Fukuda Initiative Research Unit and the Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Fukuda
Mitsunori
M
Mikoshiba
Katsuhiko
K
D000310:Adrenal Gland Neoplasms; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005934:Glucagon / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007515:Islets of Langerhans / Q000378:metabolism / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D010673:Pheochromocytoma; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013572:Synaptic Vesicles / Q000502:physiology; D050857:Synaptotagmins; D014162:Transfection
D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C494297:Syt5 protein, rat; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins; D005934:Glucagon; D002118:Calcium