ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway.
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.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.Show MeSH
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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.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.