title: Genetic analysis of yeast Sec24p mutants suggests cargo binding is not co-operative during ER export.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Roy
authors.initials: R
authors.lastname: Buchanan
authors.forename: Andrew
authors.initials: A
authors.lastname: Kaufman
authors.forename: Leslie
authors.initials: L
authors.lastname: Kung-Tran
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Miller
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D050683:R-SNARE Proteins; C118289:SEC24 protein, S cerevisiae; C083574:SMY2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C106909:Sec22 protein, S cerevisiae; D033921:Vesicular Transport Proteins