SNARE chaperone Sly1 directly mediates close-range vesicle tethering

The essential Golgi protein Sly1 is a member of the SM (Sec1/mammalian Unc-18) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory, but also acts as a positive effector. An ALPS (amphipathic lipid packing sensors)-like amphipathic helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Methods
SGA analysis

A query strain (AMY2443) was constructed in the Y9205 genetic background (Tong and Boone, 2005), with sly1∆loop and a linked nourseothricin (NAT) marker integrated through allelic replacement at the native SLY1 locus. This query strain was crossed to the MAT a haploid deletion and DAmP libraries, where each individual genetic perturbation is marked with a KAN resistance marker (Breslow et al., 2008; Tong and Boone, 2005). Diploids were selected by robotic pinning (Singer RoToR) onto YPD + 100 mg/L clonNAT + 200 mg/L G418, then induced to sporulate by pinning to sporulation medium (20g/L agar, 10g/L potassium acetate, 1g/L yeast extract, 0.5g/L glucose, 0.1g/L amino acid supplement [2g histidine, 10g leucine, 2g lysine, 2g uracil]) and growth at room temperature for 5 days. Spores were subsequently pinned to haploid selection medium (SD -His/Arg/Lys + 50 mg/L canavanine + 50 mg/L thialysine) and MAT a meiotic progeny grown for 2 days at 25º C. This haploid selection step was repeated, and the resulting colonies imaged using a Phenobooth (Singer) imaging system. These colonies encompass all potential meiotic progeny and serve as the control strains for phenotypic normalization. Haploid double mutants carrying both the KAN deletion allele and the sly1∆loop::NAT allele were selected by pinning meiotic progeny to double selection medium (SD/MSG -His/Arg/Lys + 50mg/L canavanine + 50 mg/L thialysine +100 mg/L clonNAT + 200 mg/L G418). After 2 days of growth at 25º C, this selection step was repeated and duplicate plates incubated at either 30º C or 37º C. Plates were imaged using the Phenobooth system, and colony size differences calculated using PhenoSuite software and web app (https://singerinstruments.shinyapps.io/phenobooth/).

Gene Ongology analysis of sly1-∆loop interacting genes with log ratio score ≤0.5

http://geneontology.org

Analysis Type:
PANTHER Overrepresentation Test (Released 20190711)

Annotation Version and Release Date:
PANTHER version 14.1 Released 2019-03-12

Analyzed List:
sly1-∆loop (Saccharomyces cerevisiae)

Reference List:
Saccharomyces cerevisiae (all genes in database)

Test Type:
FISHER

Correction:
BONFERRONI

Bonferroni count:
732