Characterization of yeast clathrin and anticlathrin heavy‐chain monoclonal antibodies

Sandra K. Lemmon, Vance P. Lemmon, Elizabeth W. Jones

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

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 approximately 180,000 Mr heavy chains and a single light-chain type of approximately 38,000 Mr 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 Mr, which represent the major portion of the heavy-chain arm, and a polypeptide of approximately 43,000 Mr, 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.

Original languageEnglish (US)
Pages (from-to)329-340
Number of pages12
JournalJournal of cellular biochemistry
Volume36
Issue number4
DOIs
StatePublished - Apr 1988
Externally publishedYes

Keywords

  • Saccharomyces cerevisiae
  • clathrin
  • coated vesicles
  • proteolytic digestion
  • reassembly

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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