COPI 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-hyb rid system, we characterize the domain structure of the alpha-, beta'-, eps ilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and iden tify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the beta-, gamma-, delta-, zeta -COP subcomplex and AP adaptor complexes is related. Through in vivo analys is of alpha-COP truncation mutants, we characterize distinct functional dom ains on alpha-COP, Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependen t trafficking. The last similar to 170 amino acids of alpha-COP are also no nessential for cell viability, but required for epsilon-COP incorporation i nto coatomer and maintainance of normal epsilon-COP levels. Further, we dem onstrate navel direct interactions of coatomer subunits with regulatory pro teins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (G AP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.