Architecture of coatomer: Molecular characterization of δ-COP and protein interactions within the complex

Abstract

Coatomer is a cytosolic protein complex that forms the coat of COP 1- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] α- ζ), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine α-COP, the 135-kD subunit of coatomer as well as δ-COP, the 57-kD subunit and have identified a yeast homolog of δ-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. δ-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP 1-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the δ-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two hybrid system in a complete COP screen, showed physical interactions between α- and εCOPs and between β-COPs. Moreover, the two-hybrid system indicates interactions between γ- and ζ-COPs as well as between α- and β'-COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.