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

AUTOR(ES)

Eugster, Anne

FONTE

Oxford University Press

RESUMO

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 α-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 non-essential 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.

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