Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

Abstract

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1⌬ growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1⌬ mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1⌬ vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1⌬ strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1⌬-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1⌬ strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.

Natamycin Inhibits Vacuole Fusion at the Priming Phase via a Specific Interaction with Ergosterol

“…Ergosterol is known to be important during fusion and fission processes, including vacuole fusion (12,19,31). To determine if natamycin was able to act on these processes via its specific interaction with ergosterol, the effects of this antibiotic on the fusion of isolated yeast vacuoles were studied using a content mixing assay (15,37).…”

“…Ergosterol has been shown to be required for the fusion of vacuoles, indicating the importance of ergosterol in vacuolar fusion in yeast (21,31). Here we have used this model system to decipher the mode of action of natamycin.…”

Phosphorylation-dependent Activation of Peroxisome Proliferator Protein PEX11 Controls Peroxisome Abundance

“…Only antibody Q8 was found to immunoprecipitate Pex11p from enriched peroxisomal fractions. Pot1p-, Pex3p-, and actin-specific antibodies have been described (4,24,25).…”

The Na+/H+ exchanger Nhx1p regulates the initiation of Saccharomyces cerevisiae vacuole fusion

“…Antibodies against actin, Nyv1p, Pep4p, Pho8p, Sec17p, Sec18p, Vam3p, Vam7p, Vps11p, Vps16p, Vps33p, Vps39p and Vps41p were described previously Mayer and Wickner, 1997;Nichols et al, 1997;Seals et al, 2000;Haas et al, 1995;Tedrick et al, 2004;. Anti-Vph1p monoclonal antibody was purchased from MitoSciences (Eugene, OR, USA).…”