Stimulus-induced exocytosis plays a key role in homeostasis and is involved in glandular, synaptic and other secretory systems. Trichocyst release in Paramecium tetraurelia is being studied as a model of stimulus-induced exocytosis. This phenomenon is Ca++ mediated and involves expansion of the secretion granule and membrane fusion. Recently, we have shown that dephosphorylation of a 65,000 Mr phosphoprotein (65kPP) is correlated with one or more of these signatures of secretion. Secretory mutants are available in this cell. In this proposal, we aim to extend our original observations to show that the protein dephosphorylation is involved in one of the regulatory steps between stimulus and secretion in this cell. We then will examine additional mutant strains blocked at different points along the secretory pathway to specify further the part of the pathway correlated with dephosphorylation. Additional secretagogues that may differentiate between content (tmx) expansion and membrane fusion in the mutants will also be studied. Using these secretagogues, we will follow the time course of dephosphorylation and rephosphorylation after a stimulus. Wild type (wt) and mutant cells will be exposed to calmodulin (CaM) antagonists that inhibit stimulus-induced secretion to test whether dephosphorylation is concurrently inhibited. We also hope to obtain a well characterized cell fraction from wt cells that shows an enrichment of a 65kPP, and then to define conditions for the cell fraction under which the protein can be dephosphorylated and rephosphorylated in vitro. Our ultimate objective is a description of the precise sequence of protein modifications and morphological changes that occur locally in the secretory organelle and the cell membrane during stimulus-secretion coupling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032767-03
Application #
3281864
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1983-12-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
3
Fiscal Year
1986
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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