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.
Eugster
A
A
Department of Clinical Biochemistry, University of Cambridge, Hills Road, Cambridge CB2 2XY, UK
Frigerio
G
G
Dale
M
M
Duden
R
R
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D001665:Binding Sites; D001692:Biological Transport; D020756:Coatomer Protein / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D015801:Helminth Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; D015801:Helminth Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C400683:che-2 protein, C elegans; D020727:ADP-Ribosylation Factors