ER-Golgi transport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit, Sec24p.
Selective cargo capture into ER-derived vesicles is driven by the Sec24p subunit of the COPII coat, which contains at least three independent cargo-binding sites. One of these, the "A-site," interacts with a NPF motif found on the SNARE, Sed5p. We have characterized the Sec24p-Sed5p interaction through mutation of the putative ER export motifs of Sed5p and the cargo-binding A-site of Sec24p. Mutational analysis of Sed5p suggests that the NPF motif is the dominant ER export signal. Mutation of the NPF binding pocket on Sec24p led to a dramatic reduction in the capture of Sed5p into COPII vesicles, whereas packaging of other ER-Golgi SNAREs was normal. Of all the cargoes tested, only Sed5p was depleted in vesicles made with Sec24p A-site mutants. Surprisingly, vesicles generated with the mutant Sec24p were unable to fuse with the Golgi apparatus. This inability to fuse was not the result of the lack of Sed5p, because vesicles specifically depleted of Sed5p generated by antibody inhibition targeted and fused normally. We propose that the A-site of Sec24p is a multipurpose cargo-binding site that must recognize additional unidentified cargo proteins, at least one of which is essential at a late stage of vesicle fusion.
Miller
Elizabeth A
EA
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California-Berkeley, Berkeley, CA 94702, USA.
Liu
Yiting
Y
Barlowe
Charles
C
Schekman
Randy
R
D001692:Biological Transport; D022181:COP-Coated Vesicles / Q000378:metabolism*; D049109:Cell Proliferation; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C077554:Sed5 protein, S cerevisiae