In vitro assembly and disassembly of coatomer

Abstract

Coatomer, a complex of seven proteins, is the major component of the non- clathrin (COP I) membrane coat. We report here the first system to reversibly disassemble and reassemble this complex in vitro. Coatomer disassembles at high salt concentrations and reassembles when returned to a more physiological buffer. Using this system, we show that α-, β', and ε-COP interact directly and that γ-COP interacts with ξ-COP. A partial complex comprising α-, β'-, and ε-COP, obtained after coatomer disassembly, can bind to membranes in vitro. This binding is, at least in part, mediated by interactions with cytoplasmic KKXX motifs of proteins normally retained in or retrieved to the endoplasmic reticulum. Using coatomer disassembly and epitope-specific antibodies, we also demonstrate that the N- and C-terminal domains of β-COP are buried within the native coatomer complex. These results provide the first insights into how the coatomer is structured.