SNAREs support atlastin-mediated homotypic ER fusion in <i>Saccharomyces cerevisiae</i>

Lee, Miriam Chang Yi; Ko, Young-Joon; Moon, Yeojin; Han, Minsoo; Kim, Hyung‐Wook; Lee, Sung Haeng; Kang, Kyung-In; Jun, Youngsoo

doi:10.1083/jcb.201501043

Cited by 19 publications

(43 citation statements)

References 74 publications

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“…The yeast strains used in this study are listed in Supplemental Table S1. The budding yeast Saccharomyces cerevisiae strains BJ-Gluc1 and BJ-Gluc2 were generated as previously described (17). SEY1, ERG2, ERG3, ERG4, ERG5, or ERG6 was deleted in these strains by PCRmediated gene disruption using the KanMX4 gene (24).…”

Section: Construction Of Yeast Strainsmentioning

confidence: 99%

“…GST-Sey1p-CD was prepared as previously described (17). MBP-Erg11p-CD was produced in Escherichia coli Rosette 2 (lDE3) cells in LB medium containing ampicillin by induction with 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 10 h at 16°C.…”

Section: Preparation Of Proteins and Antibodiesmentioning

confidence: 99%

“…After the beads were washed with wash buffer [20 mM Na-phosphate (pH 7.0), 500 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.4% Triton X-100] containing 20 mM imidazole, his 6 -Sey1p and his 6 -Sey1p-W685A/F707A were eluted with wash buffer containing 250 mM imidazole and dialyzed against RB150 buffer [20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 10% glycerol, and 1 mM EDTA] containing 0.4% Triton X-100. Affinity-purified anti-Sey1p and anti-Sec22p rabbit antisera were previously described (17). Anti-Yet3p and anti-Kar2p rabbit antisera were generous gifts from Dr. Charles Barlowe (Dartmouth Geisel School of Medicine, Hanover, NH, USA).…”

Section: Preparation Of Proteins and Antibodiesmentioning

confidence: 99%

“…Thereafter, GTP hydrolysis tightens membrane docking and subsequently induces dramatic conformational changes in atlastins, which causes lipid bilayer merger to complete membrane fusion between ER tubules (13,14). Despite intensive studies over the past decade, only a few factors involved in this process, other than atlastins, have been discovered (16,17). It remains largely unknown how the lipid composition of ER tubules affects atlastinmediated homotypic ER membrane fusion.…”

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confidence: 99%

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Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion inSaccharomyces cerevisiae

et al. 2018

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Sterols play critical roles in various membrane fusion events, including soluble NSF attachment protein receptor‐mediated membrane fusion, mainly by modulating the physical properties of biologic membranes; however, it remains unclear whether they also function in atlastin‐mediated endoplasmic reticulum (ER) membrane fusion. Although ergosterol, the major sterol in yeast, is essential for fusion of Sey1p (yeast atlastin)‐containing liposomes with an ER‐mimicking lipid composition, fusion of phosphatidylcholine/phosphatidylserine liposomes does not require sterols. Here, we examined whether sterols are important for Sey1p‐mediated ER fusion in Saccharomyces cerevisiae using an in vitro ER fusion assay with isolated yeast ER microsomes. Ergosterol‐specific ligands inhibited microsome fusion, indicating that ergosterol is critical for ER fusion. However, microsomes isolated from yeast strains lacking genes that encode enzymes involved in synthesis of ergosterol from lanosterol still fused, suggesting that other sterols can replace ergosterol and support Sey1p‐mediated ER fusion. Importantly, disruption of sterol‐binding motifs in the transmembrane regions of Sey1p markedly reduced ER fusion. Sey1p physically interacted with Erg11p and Erg4p, which function in ergosterol biosynthesis, suggesting that Sey1p recruits ergosterol‐synthesizing enzymes to fusion sites and thereby enriches ergosterol, which, in turn, may recruit more Sey1p. This positive feedback loop may facilitate ER membrane fusion by concentrating fusion factors at fusion sites.—Lee, M., Moon, Y., Lee, S., Lee, C., Jun, Y. Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae. FASEB J. 33, 3590–3600 (2019). http://www.fasebj.org

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Section: Construction Of Yeast Strainsmentioning

confidence: 99%

Section: Preparation Of Proteins and Antibodiesmentioning

confidence: 99%

Section: Preparation Of Proteins and Antibodiesmentioning

confidence: 99%

mentioning

confidence: 99%

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Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion inSaccharomyces cerevisiae

et al. 2018

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“…The copies of R- and Q- SNAREs per RPL are 270 and 96 respectively, according to the published calculation method (7). Typically, 65 - 85% of the SNAREs face outward (sensitive to trypsin digestion (56)).…”

Section: Methodsmentioning

confidence: 99%

Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

Arnold

Adhikari

Kang

et al. 2017

Biochemical Journal

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Sec1-Munc18 (SM) proteins cooperate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

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Closing a gap in the nuclear envelope

Vietri

Stenmark

Campsteijn

2016

Current Opinion in Cell Biology

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SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

Abstract: A quantitative in vitro assay based on isolated yeast microsomes reveals that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

Cited by 19 publications

References 74 publications

Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion inSaccharomyces cerevisiae

Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion inSaccharomyces cerevisiae

Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

Closing a gap in the nuclear envelope

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