Secretion in AtT-20 cells stably transfected with soluble synaptotagmins.
Synaptotagmin (p65) is an integral membrane secretory vesicle-specific protein with two cytoplasmic repeats homologous to the C2 regulatory domain of protein kinase C. Synaptotagmin has been implicated in the regulation of neurotransmitter release from nerve growth factor-differentiated PC12 cells and from synapses in Drosophila, Caenorhabditis elegans, and squid. To address the function of synaptotagmin in endocrine cells, fragments of rat synaptotagmin I were stably expressed in the mouse anterior pituitary cell line AtT-20. The logic of these experiments is that the fragments may interfere with the endogenous synaptotagmin machinery, thus producing a dominant-negative phenotype. Transfected cells expressed the expected fragments that were comprised of either the first C2 repeat, the second C2 repeat, or the entire cytoplasmic domain. The fragments were localized to both soluble and membrane-associated cellular fractions, despite the absence of the transmembrane domain. The second C2 repeat was shown to coimmunoprecipitate with endogenous synaptotagmin, suggesting that protein-protein interactions are mediating the membrane association of the fragments. These fragments had no effect on the targeting of regulated secretory vesicles or on regulated secretion as assayed by the release of ACTH and [3H]choline. Constitutive secretion assayed by the release of glycosaminoglycan side chains was also unaffected, as was the endocytic pathway monitored by the uptake and clearance of transferrin. These data suggest either the existence of a redundant pathway in secretion or that regulated membrane traffic in endocrine cells does not require synaptotagmin.
Wendland
B
B
Howard Hughes Medical Institute, Stanford University, California 94305.
Scheller
R H
RH
D000236:Adenoma / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000473:pathology; D011388:Prolactin / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins; D014162:Transfection; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D011388:Prolactin