Many eukaryotic secretory proteins are selected for export from the endoplasmic reticulum (ER) through their interaction with the Sec24p subunit of the coat protein II (COPII) coat. Three distinct cargo-binding sites on yeast Sec24p have been described by biochemical, genetic and structural studies. Each site recognizes a limited set of peptide motifs or a folded structural domain, however, the breadth of cargo recognized by a given site and the dynamics of cargo engagement remain poorly understood. We aimed to gain further insight into the broader molecular function of one of these cargo-binding sites using a non-biased genetic approach. We exploited the in vivo lethality associated with mutation of the Sec24p B-site to identify genes that suppress this phenotype when overexpressed. We identified SMY2 as a general suppressor that rescued multiple defects in Sec24p, and SEC22 as a specific suppressor of two adjacent cargo-binding sites, raising the possibility of allosteric regulation of these domains. We generated a novel set of mutations in Sec24p that distinguish these two sites and examined the ability of Sec22p to rescue these mutations. Our findings suggest that co-operativity does not influence cargo capture at these sites, and that Sec22p rescue occurs via its function as a retrograde SNARE.

• PMID: 20477990
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Journal Article | Research Support, N.I.H., Extramural

Authors

Buchanan R, Kaufman A, Kung-Tran L, Miller EA

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