Mutational analysis of betaCOP (Sec26p) identifies an appendage domain critical for function.
BACKGROUND
The appendage domain of the gammaCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the gammaCOP appendage domain and an equivalent region on betaCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in betaCOP.


RESULTS
Sequence comparisons in combination with structural prediction tools show that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Deletion of the appendage domain of Sec26p results in inviability in yeast, over-expression of the deletion construct is dominant negative and mutagenesis of this region identifies residues critical for function. The ArfGAP Glo3p was identified via suppression screening as a potential downstream modulator of Sec26p in a manner that is independent of the GAP activity of Glo3p but requires the presence of the COOH-terminal ISS motifs.


CONCLUSIONS
Together, these results indicate an essential function for the predicted betaCOP appendage and suggest that both COPI appendages perform a biologically active regulatory role with a structure related to adaptin-family appendage domains.
DeRegis
Carol J
CJ
Graduate Program in Comparative Biomedical Sciences, Cornell University, Ithaca NY 14853, USA. cjd37@cornell.edu
Rahl
Peter B
PB
Hoffman
Gregory R
GR
Cerione
Richard A
RA
Collins
Ruth N
RN
D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D022181:COP-Coated Vesicles / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D020756:Coatomer Protein / Q000737:chemistry* / Q000235:genetics*; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; C514182:Glo3 protein, S cerevisiae; C089601:SEC26 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins