Identification of a distinct 9S form of soluble clathrin in cultured cells and tissues

Abstract

We have used a monoclonal antibody (CHC5.9) to identify clathrin (Mr 180,000; 'heavy chain') in coated vesicles, triskelion structures prepared in vitro and in high-speed supernatants (HSS) of cell homogenates from a variety of tissues and species (e.g., brain and liver from rat, cow and man; Xenopus ovaries). HSS proteins were subjected to sucrose density gradient centrifugation and gel filtration, and the fractions obtained were assayed for clathrin by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting. The native soluble clathrin identified in such fractions was indistinguishable from triskelions produced in vitro from purified bovine brain clathrin by several criteria, e.g. by its sedimentation coefficient (9S) and elution profile on gel filtration using Sephacryl S 300. No other major forms of soluble clathrin were detected. The results indicate that cells contain a soluble pool of clathrin and that the predominant molecular form of this soluble clathrin has properties similar to those of the triskelion obtained by dissociation studies in vitro. We hypothesize that this distinct 9S form represents a major oligomeric subunit involved in assembly and disassembly of clathrin polyhedron coats in the living cell.