COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the α-, β'-, ε-COP and β-, γ-, δ-, ζ-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the β-, γ-, δ-, ζ-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of α-COP truncation mutants, we characterize distinct functional domains on a-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent trafficking. The last ~170 amino acids of α-COP are also nonessential for cell viability, but required for ε-COP incorporation into coatomer and maintainance of normal ε-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: β'- and γ-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and β- and ε-COP interact with ARF-GTP, Glo3p also interacts with intact coatomer in vitro.