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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.

Pubmed ID: 10921873

Authors

  • Eugster A
  • Frigerio G
  • Dale M
  • Duden R

Journal

The EMBO journal

Publication Data

August 1, 2000

Associated Grants

None

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