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ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway.

Rogers JV, McMahon C, Baryshnikova A, Hughson FM, Rose MD - Mol. Biol. Cell (2014)

Bottom Line: However, an alternative explanation--that the observed phenotypes arose from perturbed vesicle trafficking--could not be ruled out.In contrast, cytosolic coat protein I (COPI) vesicle coat mutations in sey1Δ cells caused no synthetic defects, excluding perturbed retrograde trafficking as a cause for the previously observed synthetic defects.We conclude that the ER SNAREs and the Dsl1 complex directly mediate Sey1p-independent ER-ER fusion and that, in the absence of both pathways, cell viability depends upon membrane curvature-promoting reticulons.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.

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SNARE-mediated homotypic ER fusion requires the entire Dsl1 complex but not the COPI coat. (A) Growth assays of wild type (MY14289), sey1Δ (MY14291), sec39-1 (MY14293), and sey1Δ sec39-1 (MY14296) grown for 2 d at 30°C. (B) Top, wild type (MY14653), sey1Δ (MY14655), sec27-1 (MY14657), and sey1Δ sec27-1 (MY14659) grown for 3 d at 23°C and 2 d at 30°C. Bottom, wild type (MY14653), sey1Δ (MY14655), ret2-1 (MY14661), and sey1Δ ret2-1 (MY14663) grown at the indicated temperatures for 2 d. (C) Indicated genotypes derived from parent MY15008 grown for 2 d at 27°C. (D) Indicated genotypes with plasmid pRY270 (dsl1-A533D) or pRY261 (dsl1-L55E/L58D). Top, wild type (MY14365), sey1Δ (MY14369), dsl1Δ (MY14373), and sey1Δ dsl1Δ (MY14377). Bottom, wild type (MY14384), sey1Δ (MY14388), dsl1Δ (MY14392), and sey1Δ dsl1Δ (MY14396).
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Figure 3: SNARE-mediated homotypic ER fusion requires the entire Dsl1 complex but not the COPI coat. (A) Growth assays of wild type (MY14289), sey1Δ (MY14291), sec39-1 (MY14293), and sey1Δ sec39-1 (MY14296) grown for 2 d at 30°C. (B) Top, wild type (MY14653), sey1Δ (MY14655), sec27-1 (MY14657), and sey1Δ sec27-1 (MY14659) grown for 3 d at 23°C and 2 d at 30°C. Bottom, wild type (MY14653), sey1Δ (MY14655), ret2-1 (MY14661), and sey1Δ ret2-1 (MY14663) grown at the indicated temperatures for 2 d. (C) Indicated genotypes derived from parent MY15008 grown for 2 d at 27°C. (D) Indicated genotypes with plasmid pRY270 (dsl1-A533D) or pRY261 (dsl1-L55E/L58D). Top, wild type (MY14365), sey1Δ (MY14369), dsl1Δ (MY14373), and sey1Δ dsl1Δ (MY14377). Bottom, wild type (MY14384), sey1Δ (MY14388), dsl1Δ (MY14392), and sey1Δ dsl1Δ (MY14396).

Mentions: We manually verified the most important SGA results and checked for synthetic growth defects between sey1Δ and candidates that were missing from the SGA arrays. We confirmed the synthetic growth defect in sey1Δ sec39-1 mutants (Figure 3A) and the lack of a COPI interaction (no growth defects in sey1Δ sec27-1, sey1Δ ret2-1, and sey1Δ cop1-1 mutants; Figure 3B and Supplemental Figure S1A). We also tested the third and final subunit of the Dsl1 complex, Tip20p, which was missing from the SGA analysis; sey1Δ tip20-5 mutants exhibited strong synthetic growth defects, implicating the entire Dsl1 complex in ER–ER fusion (Figure 3C).


ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway.

Rogers JV, McMahon C, Baryshnikova A, Hughson FM, Rose MD - Mol. Biol. Cell (2014)

SNARE-mediated homotypic ER fusion requires the entire Dsl1 complex but not the COPI coat. (A) Growth assays of wild type (MY14289), sey1Δ (MY14291), sec39-1 (MY14293), and sey1Δ sec39-1 (MY14296) grown for 2 d at 30°C. (B) Top, wild type (MY14653), sey1Δ (MY14655), sec27-1 (MY14657), and sey1Δ sec27-1 (MY14659) grown for 3 d at 23°C and 2 d at 30°C. Bottom, wild type (MY14653), sey1Δ (MY14655), ret2-1 (MY14661), and sey1Δ ret2-1 (MY14663) grown at the indicated temperatures for 2 d. (C) Indicated genotypes derived from parent MY15008 grown for 2 d at 27°C. (D) Indicated genotypes with plasmid pRY270 (dsl1-A533D) or pRY261 (dsl1-L55E/L58D). Top, wild type (MY14365), sey1Δ (MY14369), dsl1Δ (MY14373), and sey1Δ dsl1Δ (MY14377). Bottom, wild type (MY14384), sey1Δ (MY14388), dsl1Δ (MY14392), and sey1Δ dsl1Δ (MY14396).
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Figure 3: SNARE-mediated homotypic ER fusion requires the entire Dsl1 complex but not the COPI coat. (A) Growth assays of wild type (MY14289), sey1Δ (MY14291), sec39-1 (MY14293), and sey1Δ sec39-1 (MY14296) grown for 2 d at 30°C. (B) Top, wild type (MY14653), sey1Δ (MY14655), sec27-1 (MY14657), and sey1Δ sec27-1 (MY14659) grown for 3 d at 23°C and 2 d at 30°C. Bottom, wild type (MY14653), sey1Δ (MY14655), ret2-1 (MY14661), and sey1Δ ret2-1 (MY14663) grown at the indicated temperatures for 2 d. (C) Indicated genotypes derived from parent MY15008 grown for 2 d at 27°C. (D) Indicated genotypes with plasmid pRY270 (dsl1-A533D) or pRY261 (dsl1-L55E/L58D). Top, wild type (MY14365), sey1Δ (MY14369), dsl1Δ (MY14373), and sey1Δ dsl1Δ (MY14377). Bottom, wild type (MY14384), sey1Δ (MY14388), dsl1Δ (MY14392), and sey1Δ dsl1Δ (MY14396).
Mentions: We manually verified the most important SGA results and checked for synthetic growth defects between sey1Δ and candidates that were missing from the SGA arrays. We confirmed the synthetic growth defect in sey1Δ sec39-1 mutants (Figure 3A) and the lack of a COPI interaction (no growth defects in sey1Δ sec27-1, sey1Δ ret2-1, and sey1Δ cop1-1 mutants; Figure 3B and Supplemental Figure S1A). We also tested the third and final subunit of the Dsl1 complex, Tip20p, which was missing from the SGA analysis; sey1Δ tip20-5 mutants exhibited strong synthetic growth defects, implicating the entire Dsl1 complex in ER–ER fusion (Figure 3C).

Bottom Line: However, an alternative explanation--that the observed phenotypes arose from perturbed vesicle trafficking--could not be ruled out.In contrast, cytosolic coat protein I (COPI) vesicle coat mutations in sey1Δ cells caused no synthetic defects, excluding perturbed retrograde trafficking as a cause for the previously observed synthetic defects.We conclude that the ER SNAREs and the Dsl1 complex directly mediate Sey1p-independent ER-ER fusion and that, in the absence of both pathways, cell viability depends upon membrane curvature-promoting reticulons.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.

Show MeSH
Related in: MedlinePlus