This proposal concerns detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in eukaryotic cells. There are least three cellular compartments involved in the process of signal transduction in exocytosis: (1) the plasma membrane which contains secretagogue receptors and other transmembrane proteins (2) the cytoplasm particularly in the region between the cell and secretory vesicle membrane, where molecules may influence interactions of the membranes and (3) the secretory vesicle itself. The properties of the vesicle content and its membrane are also crucial to the interactions that finally accompany product release. The details of signal transduction during exocytosis will be studied in Paramecium tetraurelia. Gilligan and Satir demonstrated that when normal exocytosis was induced in wild type cells a dephosphorylation could be detected in a number of Paramecium phosphoproteins, most prominently in a Mr 63kDa species. Preincubation of cells in high Mg2+ (no added Ca2+) inhibited both exocytosis and dephosphorylation in response to secretagogue. Further, a mutant nd9, when grown at 18 degree C (permissive temperature), had the normal rosettes at the secretory site, secreted normally and dephosphorylated the 63kDa polypeptide in response to secretagogue, but when grown at 27 degree C (non-permissive temperature) where rosettes were not assembled, did not secrete nor dephosphorylate the 63kDa polypeptide in response to the secretagogue. We have succeeded in purifying the 63kDa protein and have made a polyclonal antibody against it. We intend to investigate its intracellular localization, exploring the possibility that the protein becomes an integral part of the cytoskeleton in the rosette region upon dephosphorylation. Using Western blot analysis with the purified antibody on other cells and mammalian tissues, we will examine whether the 63kDa protein may be of general significance in other secretory processes. We shall attempt to define, characterize and localize the cellular phosphatase(s) and kinase(s) that act on the 63kDa protein. In addition, the influx of extracellular Ca2+ after appropriate stimulation appears to be mediated by specific exocytic Ca2+ channels, separate and distinct from the well known voltage dependent ciliary Ca2+ channels. We intend to characterize and localize these channels more completely.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032767-06
Application #
3281866
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1983-12-01
Project End
1992-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Subramanian, S V; Satir, B H (1992) Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium. Proc Natl Acad Sci U S A 89:11297-301
Ding, Y; Ron, A; Satir, B H (1991) A potential mucus precursor in Tetrahymena wild type and mutant cells. J Protozool 38:613-23
Satir, B H; Srisomsap, C; Reichman, M et al. (1990) Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver. J Cell Biol 111:901-7
Bleyman, L K; Satir, B H (1990) Chromosomal localization of an exocytosis mutant in Tetrahymena thermophila. J Protozool 37:471-2
Busch, G R; Satir, B H (1989) The secretory vesicle in living Paramecium is acidic. J Cell Sci 92 ( Pt 2):197-203
Satir, B H; Busch, G; Vuoso, A et al. (1988) Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium. J Cell Biochem 36:429-43
Murtaugh, T J; Gilligan, D M; Satir, B H (1987) Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium. J Biol Chem 262:15734-9
Satir, B H; Reichman, M; Orias, E (1986) Conjugation rescue of an exocytosis-competent membrane microdomain in Tetrahymena thermophila mutants. Proc Natl Acad Sci U S A 83:8221-5
Maihle, N J; Satir, B H (1986) Protein secretion in Tetrahymena thermophila. Characterization of the major proteinaceous secretory proteins. J Biol Chem 261:7566-70
Maihle, N J; Satir, B H (1986) Identification of a biochemical marker for the secretory pathway in Tetrahymena thermophila. J Cell Biochem 31:195-202

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