SNARE chaperone Sly1 directly mediates close-range vesicle tethering
Data files
Feb 27, 2024 version files 1.01 MB
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Duan_sly1_SGA_SuppDataset_dryadVers2.xlsx
1.01 MB
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README.md
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Abstract
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.
README: Yeast Sly1 SGA, BioGrid, and Gene Ontology Supplemental Dataset
https://doi.org/10.5061/dryad.dr7sqvb5b
Description of the data and file structure:
To gain genome-scale insight into the sly1∆loop allele’s loss of function, we used synthetic genome array (SGA) analysis. SGA measures the synthetic sickness or rescue (suppression) of a query allele versus a genome-scale collection of loss-of-function alleles (Tong and Boone, 2005). The sly1∆loop allele was knocked into the genomic SLY1 locus. The SGA data from this analysis was then aligned with the BioGRID dataset.
Contents of the dataset:
sly1∆loop SGA contains the raw SGA dataset. The SGA score algorithm processes raw colony size data, normalizes them for a series of experimental systematic effects and calculates a quantitative genetic interaction score.
LogRatios indicates the log-transformed ratio of the growth of the indicated double mutant to the growth of the single mutant with the indicated query allele.
ZScores is a unitless indication of divergence from the mean. Z=1 corresponds to 1 standard deviation from the dataset mean.
UID_miller is a unique identifier code used within the Miller laboratory.
Run indicates the experiment number.
PlateA indicates the plate index number.
Type indicates experiment or control.
RowA indicates the row index letter for the specified PlateA.
ColA indicates the column index number for the specified plate A.
Exp.Size Colony area measured in pixels.
Exp.Circularity Quantitative method to identify the roundness of the colony in a 2-dimensional plane or how close the colony is to a true circle (C=1).
Exp.NSize Colony size after normalization with the rest of the plate. This is done to control systemic variability when comparing colonies from different screens and different locations on the plate.
RowNumeric is the conversion of the alphabetical rows in RowB converted to numbers. A=1, B=2, etc.
Ctrl.Size Size of the control strain colonies in pixels.
Ctrl.NSize Size of the control strain colonies normalized.
PlateB indicates the plate index number.
RowB indicates the row index letter for the specified PlateB.
ColB indicates the column index number for specified PlateB.
GO analysis contains the parameters and outputs for Gene Ontology analysis of the SGA dataset.
sly1 Biogrid includes all genes reported to have negative interactions with SLY1 as of 2019-11 (at www.thebiogrid.org).
Columns K and L are gene synonyms for the bait and prey (A and B). Many genes don’t have alternative names so the cells are left blank.
Author refers to the literature reference cited for the entry in the BioGrid data repository.
sly1∆loop SGA and Biogrid compares scores for genes found in both our SGA interaction dataset and the BioGRID dataset.
UID_miller is a unique identifier code used within the Miller laboratory.
LogRatios indicates the log-transformed ratio of the growth of the indicated double mutant to the growth of the single mutant with the indicated query allele.
ZScores is a unitless indication of divergence from the mean. Z=1 corresponds to 1 standard deviation from the dataset mean.
uid_biogrid is a unique record identifier used by the BioGrid data repository.
SGA Score is a unitless genetic interaction score.
NA = indicates that tests that were done and reported as genetic interactions but NOT quantified with an SGA score.
This Methods section is duplicated and slightly modified from the JCB Article (10.1083/jcb.202001032).
Sharing/Access information
Links to other publicly accessible locations of the data:
- https://doi.org/10.1101/2020.01.16.906719
- 10.1083/jcb.202001032
Data was derived from the following sources:
- Synthetic genome array (SGA) analysis
- Gene Ontology http://geneontology.org
- BioGRID analysis https://thebiogrid.org/
Code/Software
Plates were imaged using the Phenobooth system, and colony size differences calculated using PhenoSuite software and web app (https://singerinstruments.shinyapps.io/phenobooth/).
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 |