Mutational analysis of betaCOPI (Sec26p) identifies an appendage domain critical for function

Graduate Program in Comparative Biomedical Sciences, Cornell University, Ithaca NY 14853, USA.
BMC Cell Biology (Impact Factor: 2.34). 02/2008; 9(1):3. DOI: 10.1186/1471-2121-9-3
Source: PubMed


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.
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.
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.

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    • "The Saccharomyces cerevisiae strains used in this study are listed in Table S1 and were created using standard manipulations. For Western blot analysis, gels were transferred to PVDF Immobilon membrane (Millipore) for 2 hrs prior to probing with antibodies including anti-GFP (Chemicon) and anti-Sec26p [21]. Blots were subsequently incubated with the appropriate secondary antibody coupled to alkaline phosphatase and imaged using CDP-Star chemiluminescence reagent (Perkin Elmer) and recorded with Fuji LAS3000. "
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    • "Mutation of the ␣AP2 and ␤AP2 FW motif in combination with flanking basic residues alters binding of regulatory factors (Owen et al., 1999; Traub et al., 1999; Owen et al., 2000), suggesting the potential importance of this region in ␥COP and ␤COP. Indeed, mutation in the FW motif of Sec26p, the yeast homologue of ␤COP, causes temperaturesensitive growth defects (Hoffman et al., 2003; DeRegis et al., 2008). Thus, mutations in the FW motif of the ␥COP or ␤COP appendage domains might interfere with transport factor binding and possibly cause deficits in Golgi biogen- esis. "
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    ABSTRACT: Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex, but the mechanism is poorly understood. Modeling of the COPI subunit betaCOP based on the clathrin adaptor AP2 suggested that the betaCOP C terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of betaCOP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, betaCOP bound p115, and this depended on the betaCOP FW motif. Furthermore, the interaction depended on E(19)E(21) in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, betaCOP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in betaCOP and p115, suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.
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