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Characterization of yeast clathrin and anticlathrin heavy‐chain monoclonal antibodies
Author(s) -
Lemmon Sandra K.,
Lemmon Vance P.,
Jones Elizabeth W.
Publication year - 1988
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240360403
Subject(s) - clathrin , monoclonal antibody , yeast , immunoglobulin light chain , sepharose , biochemistry , peptide , biology , saccharomyces cerevisiae , microbiology and biotechnology , chemistry , vesicle , antibody , enzyme , membrane , immunology
Clathrin‐coated vesicles (CVs) were isolated from Saccharomyces cerevisiae by using procedures developed by Mueller and Branton [17]. Triskelions were purified from this material by extraction of CVs to release clathrin and by subsequent fractionation on Sepharose CL‐4B. Triskelions were composed of ∼ 180,000 M r heavy chains and a single light‐chain type of ∼ 38,000 M r and were able to undergo self‐assembly into polyhedral cages. Trypsin digestion of such reassembled cages showed a peptide pattern very similar to that obtained for mammalian clathrin with two fragments of 125,000 and 110,000 M r , which represent the major portion of the heavy‐chain arm, and a polypeptide of ∼ 43,000 M r , which is the presumptive terminal domain. Eight monoclonal antibodies reacting with yeast clathrin heavy chains were produced. All eight bind to the major portion of the heavy‐chain arm, and none bind to the terminal domain fragment. Peptide digestion experiments also indicated that at least three major regions on the arm are recognized by these antibodies. These will be useful in further structural and functional studies of clathrin from yeast.