POLYPEPTIDES THAT CONFER FUNCTIONAL DIVERSITY TO CLATHRIN-COATED VESICLES (MONOCLONAL ANTIBODIES).
Item
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Title
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POLYPEPTIDES THAT CONFER FUNCTIONAL DIVERSITY TO CLATHRIN-COATED VESICLES (MONOCLONAL ANTIBODIES).
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Identifier
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AAI8708296
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identifier
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8708296
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Creator
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KOHTZ, DONALD STAVE, II.
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Contributor
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Saul Puszkin
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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Four new anti-clathrin light chain mAbs are reported here: C-7H12 and C-6C1 react with both forms: C-10B2 and C-4E5 react only with the lower molecular mass form. Characterization of these mAbs indicated that the clathrin light chains contain two structural domains: a non-conserved, accessible domain that is relevant to the phosphorylation of CAP(,2) and a conserved inaccessible domain that binds clathrin.;Analysis of brain clathrin-coated vesicles (CCVs) by immunoprecipitation with anti-actin (A-7C11) or anti-tubulin (T-2H9) mAbs coupled to Sepharose 4B revealed that while all brain CCVs contain tubulin, only a distinct subpopulation is enriched in actin. Monoclonal antibodies were developed against clathrin-coated vesicle cargo molecules that are sorted into either the actin('+) or actin('-) subpopulations. The distribution of the two forms of clathrin light chains among brain clathrin-coated vesicle was also examined, using monoclonal antibodies specific for each form. Subpopulations enriched in either the higher or lower molecular mass form of clathrin light chain were immunoprecipitated with these monoclonal antibodies.;Two monoclonal antibodies are characterized that react with a previously unidentified membrane-bound polypeptide (SA). Different forms of SA protein are distinguished by one-dimensional gel electrophoresis and immunoblotting. One form (SA(,1)) is found exclusively in plasma membrane preparations, another (SA(,2)) is found in clathrin-coated vesicles and synaptic vesicles, while another (SA(,3)) is found exclusively in Golgi fractions. Analysis by in vitro translation indicates that cells producing all forms of SA protein produce only a single transcript encoding a precursor of SA(,1). Successive post-translational modifications appear to target SA protein to specific intracellular membranes.;Finally, two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant component of only neuronal clathrin-coated vesicles. This 185 kD polypeptide (NP185) binds the CCV assembly polypeptides and is biochemically, immunochemically, and functionally distinguishable from clathrin. Immunoprecipitation experiments using radioiodinated CCVs and S-8G8 revealed that NP185 can be competed off the CCV by soluble tubulin. NP185 is apparently bound to neuronal CCVs by its interaction with vesicle membrane-bound tubulin. Further analysis indicated that phosphorylation of tubulin affects its affinity for NP185.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
