ARCHITECTURE OF COATOMER - MOLECULAR CHARACTERIZATION OF DELTA-COP AND PROTEIN INTERACTIONS WITHIN THE COMPLEX

Coatomer is a cytosolic protein complex that forms the coat of COP I-c oated transport vesicles. In our attempt to analyze the physical and f unctional interactions between its seven subunits (coat proteins, [COP s] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overe xpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as d elta-COP, the 57-kD subunit and have identified a yeast homolog of del ta-COP by cDNA sequence comparison and by NH2-terminal peptide sequenc ing. delta-COP shows homologies to subunits of the clathrin adaptor co mplexes AP1 and AP2. We show that in Golgi-enriched membrane fractions , the protein is predominantly found in COPI-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as an alysis exploiting the two-hybrid system in a complete COP screen, show ed physical interactions between alpha- and epsilon-COPs and between b eta- and delta-COPs. Moreover, the two-hybrid system indicates interac tions between gamma- and zeta-COPs as well as between alpha- and beta' -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 co mplex.