DOI: 10.5061/dryad.dr7sqvb5b
title: SNARE chaperone Sly1 directly mediates close-range vesicle tethering

description: 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.
description: <strong>SGA analysis</strong> A
 query strain (AMY2443) was constructed in the Y9205 genetic background
 (Tong and Boone, 2005), with <em>sly1∆loop</em> and a linked
 nourseothricin (<em>NAT</em>) marker integrated through
 allelic replacement at the native <em>SLY1</em> locus. This
 query strain was crossed to the <em>MAT</em>
 <strong>a</strong> haploid deletion and DAmP libraries, where
 each individual genetic perturbation is marked with a
 <em>KAN</em> 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
 <em>MAT</em> <strong>a</strong> 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 <em>KAN</em> deletion allele and the
 <em>sly1∆loop</em>::<em>NAT </em>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  
description: # Yeast Sly1 SGA, BioGrid, and Gene Ontology Supplemental Dataset
 ## 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](http://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: * *
 10.1083/jcb.202001032 **Data was derived from the following sources:** *
 Synthetic genome array (SGA) analysis * Gene Ontology
 * BioGRID analysis
 ## Code/Software Plates were imaged using
 the Phenobooth system, and colony size differences calculated using
 PhenoSuite software and web app
 (