Genetic analysis of yeast Sec24p mutants suggests cargo binding is not co-operative during ER export.
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.
Buchanan
Roy
R
Department of Biological Sciences, Columbia University, New York, NY, USA.
Kaufman
Andrew
A
Kung-Tran
Leslie
L
Miller
Elizabeth A
EA
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
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