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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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ER–ER fusion is severely delayed in sey1Δ dsl1ΔE mutants. (A) Representative example of the ER–ER fusion assay. Identical genotypes were mated to each other at room temperature; MATa strains expressed mCherry-HDEL (pMR6474), MATα strains expressed cytosolic GFP (pMR3619). sey1Δ cells are shown. The first peak in mCherry transfer at 0–2 min corresponds to the transfer of cytosolic mCherry-HDEL after cell fusion. Arrowhead depicts the point of ER–ER fusion (8 min). Scale bar: 2 μm. (B) Box plot of the times required for ER–ER fusion after cell fusion. Data are pooled from at least two independent experiments for each genotype. Mean values: wild type, 4.9 min (MY14509 × MY14513, n = 16); sey1Δ, 8.8 min (MY14510 × MY14514, n = 16); dsl1ΔE, 5.3 min (MY14511 × MY14515, n = 14); and sey1Δ dsl1ΔE, 23.4 min (MY14512 × MY14516; n = 10). Each box shows the interquartile range (25–75% of the data), black bars represent the median, and outliers are shown as open circles beyond the 1.5 * interquartile range. (C) Percentage of zygotes with unfused ER (binary scoring). The data are pooled from two independent experiments; error bars show ± SE for a binomial distribution. Strains used: wild type (MY14059 × MY14009), dsl1ΔL (MY14061 × MY14013), sey1Δ (MY14063 × MY14017), sey1Δ dsl1ΔL (MY14065 × MY14021), dsl1ΔE (MY14067 × MY14025), sey1Δ dsl1ΔE (MY14071 × MY14031), and sey1Δ dsl1ΔE (MY14071) × wild type (MY14009). All MATa strains expressed GFP-HDEL (pMR6473).
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Figure 5: ER–ER fusion is severely delayed in sey1Δ dsl1ΔE mutants. (A) Representative example of the ER–ER fusion assay. Identical genotypes were mated to each other at room temperature; MATa strains expressed mCherry-HDEL (pMR6474), MATα strains expressed cytosolic GFP (pMR3619). sey1Δ cells are shown. The first peak in mCherry transfer at 0–2 min corresponds to the transfer of cytosolic mCherry-HDEL after cell fusion. Arrowhead depicts the point of ER–ER fusion (8 min). Scale bar: 2 μm. (B) Box plot of the times required for ER–ER fusion after cell fusion. Data are pooled from at least two independent experiments for each genotype. Mean values: wild type, 4.9 min (MY14509 × MY14513, n = 16); sey1Δ, 8.8 min (MY14510 × MY14514, n = 16); dsl1ΔE, 5.3 min (MY14511 × MY14515, n = 14); and sey1Δ dsl1ΔE, 23.4 min (MY14512 × MY14516; n = 10). Each box shows the interquartile range (25–75% of the data), black bars represent the median, and outliers are shown as open circles beyond the 1.5 * interquartile range. (C) Percentage of zygotes with unfused ER (binary scoring). The data are pooled from two independent experiments; error bars show ± SE for a binomial distribution. Strains used: wild type (MY14059 × MY14009), dsl1ΔL (MY14061 × MY14013), sey1Δ (MY14063 × MY14017), sey1Δ dsl1ΔL (MY14065 × MY14021), dsl1ΔE (MY14067 × MY14025), sey1Δ dsl1ΔE (MY14071 × MY14031), and sey1Δ dsl1ΔE (MY14071) × wild type (MY14009). All MATa strains expressed GFP-HDEL (pMR6473).

Mentions: Fixed-cell assays (Figure 5C) were performed as previously described (Rogers et al., 2013). Briefly, cultures were grown at 30°C to mid–log phase, and 0.5 OD600 unit of cells was mixed and mated on a 0.45-μm nitrocellulose filter (EMD Millipore, Billerica, MA) for 3 h at 23°C. Cells were then washed into 900 μl of 1× phosphate-buffered saline (PBS), and 100 μl of 20% paraformaldehyde dissolved in distilled H2O was added. Cells were fixed at room temperature for 15 min; this was followed by one wash in 1× PBS, 4′,6-diamidino-2-phenylindole staining (2 μg/ml in PBS) for 15 min, two more washes in 1× PBS, and, finally, resuspension in 100–200 μl 1× PBS. Cells were imaged on the same day. GFP-HDEL was used in the fixed-cell assay rather than mCherry-HDEL, as GFP-HDEL intensity and localization is preserved better after fixation. Imaging on the same day ensures that the membranes stay intact and GFP-HDEL does not artifactually diffuse to equilibrium. ER was scored as unfused when GFP-HDEL appeared markedly brighter in one-half of the zygote than the other.


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)

ER–ER fusion is severely delayed in sey1Δ dsl1ΔE mutants. (A) Representative example of the ER–ER fusion assay. Identical genotypes were mated to each other at room temperature; MATa strains expressed mCherry-HDEL (pMR6474), MATα strains expressed cytosolic GFP (pMR3619). sey1Δ cells are shown. The first peak in mCherry transfer at 0–2 min corresponds to the transfer of cytosolic mCherry-HDEL after cell fusion. Arrowhead depicts the point of ER–ER fusion (8 min). Scale bar: 2 μm. (B) Box plot of the times required for ER–ER fusion after cell fusion. Data are pooled from at least two independent experiments for each genotype. Mean values: wild type, 4.9 min (MY14509 × MY14513, n = 16); sey1Δ, 8.8 min (MY14510 × MY14514, n = 16); dsl1ΔE, 5.3 min (MY14511 × MY14515, n = 14); and sey1Δ dsl1ΔE, 23.4 min (MY14512 × MY14516; n = 10). Each box shows the interquartile range (25–75% of the data), black bars represent the median, and outliers are shown as open circles beyond the 1.5 * interquartile range. (C) Percentage of zygotes with unfused ER (binary scoring). The data are pooled from two independent experiments; error bars show ± SE for a binomial distribution. Strains used: wild type (MY14059 × MY14009), dsl1ΔL (MY14061 × MY14013), sey1Δ (MY14063 × MY14017), sey1Δ dsl1ΔL (MY14065 × MY14021), dsl1ΔE (MY14067 × MY14025), sey1Δ dsl1ΔE (MY14071 × MY14031), and sey1Δ dsl1ΔE (MY14071) × wild type (MY14009). All MATa strains expressed GFP-HDEL (pMR6473).
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Figure 5: ER–ER fusion is severely delayed in sey1Δ dsl1ΔE mutants. (A) Representative example of the ER–ER fusion assay. Identical genotypes were mated to each other at room temperature; MATa strains expressed mCherry-HDEL (pMR6474), MATα strains expressed cytosolic GFP (pMR3619). sey1Δ cells are shown. The first peak in mCherry transfer at 0–2 min corresponds to the transfer of cytosolic mCherry-HDEL after cell fusion. Arrowhead depicts the point of ER–ER fusion (8 min). Scale bar: 2 μm. (B) Box plot of the times required for ER–ER fusion after cell fusion. Data are pooled from at least two independent experiments for each genotype. Mean values: wild type, 4.9 min (MY14509 × MY14513, n = 16); sey1Δ, 8.8 min (MY14510 × MY14514, n = 16); dsl1ΔE, 5.3 min (MY14511 × MY14515, n = 14); and sey1Δ dsl1ΔE, 23.4 min (MY14512 × MY14516; n = 10). Each box shows the interquartile range (25–75% of the data), black bars represent the median, and outliers are shown as open circles beyond the 1.5 * interquartile range. (C) Percentage of zygotes with unfused ER (binary scoring). The data are pooled from two independent experiments; error bars show ± SE for a binomial distribution. Strains used: wild type (MY14059 × MY14009), dsl1ΔL (MY14061 × MY14013), sey1Δ (MY14063 × MY14017), sey1Δ dsl1ΔL (MY14065 × MY14021), dsl1ΔE (MY14067 × MY14025), sey1Δ dsl1ΔE (MY14071 × MY14031), and sey1Δ dsl1ΔE (MY14071) × wild type (MY14009). All MATa strains expressed GFP-HDEL (pMR6473).
Mentions: Fixed-cell assays (Figure 5C) were performed as previously described (Rogers et al., 2013). Briefly, cultures were grown at 30°C to mid–log phase, and 0.5 OD600 unit of cells was mixed and mated on a 0.45-μm nitrocellulose filter (EMD Millipore, Billerica, MA) for 3 h at 23°C. Cells were then washed into 900 μl of 1× phosphate-buffered saline (PBS), and 100 μl of 20% paraformaldehyde dissolved in distilled H2O was added. Cells were fixed at room temperature for 15 min; this was followed by one wash in 1× PBS, 4′,6-diamidino-2-phenylindole staining (2 μg/ml in PBS) for 15 min, two more washes in 1× PBS, and, finally, resuspension in 100–200 μl 1× PBS. Cells were imaged on the same day. GFP-HDEL was used in the fixed-cell assay rather than mCherry-HDEL, as GFP-HDEL intensity and localization is preserved better after fixation. Imaging on the same day ensures that the membranes stay intact and GFP-HDEL does not artifactually diffuse to equilibrium. ER was scored as unfused when GFP-HDEL appeared markedly brighter in one-half of the zygote than the other.

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