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 alpha-, beta'-, epsilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and identify 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 analysis of alpha-COP truncation mutants, we characterize distinct functional domains on alpha-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 approximately 170 amino acids of alpha-COP are also non-essential for cell viability, but required for epsilon-COP incorporation into coatomer and maintainance of normal epsilon-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.
Mesh Terms:
ADP-Ribosylation Factors, Adaptor Protein Complex alpha Subunits, Adaptor Proteins, Vesicular Transport, Binding Sites, Biological Transport, Coatomer Protein, Endoplasmic Reticulum, GTPase-Activating Proteins, Golgi Apparatus, Helminth Proteins, Membrane Proteins, Models, Molecular, Mutation, Peptide Fragments, Protein Binding, Protein Structure, Tertiary, Two-Hybrid System Techniques, Yeasts
EMBO J.
Date: Aug. 01, 2000
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