Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Abstract

ORCIDRutilio Fratti -https://orcid.org/0000-0001-9109-6666 Running title: Elo3p regulation of vacuole fusion via lipid rafts SummaryMany sphingolipids contain very-long chain fatty-acids with 26 carbons. The deletion of Elo3, the elongase that adds the final two carbons results in pleiotropic effects that negatively alter membrane fusion at the tethering and docking stages. AbstractThe role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolated from these cells displayed reduced levels of in vitro fusion, which we traced to a block in tethering and docking. We found that C26 VLCFA deficient yeast mislocalize fusion markers, and the small GTPases Rho1p and Ypt7p fail to selectively concentrate at the boundary and vertex domains of vacuoles isolated from these yeasts. Surprisingly, we only observed mild changes to the localization of other regulatory lipids, but membrane fluidity and solubility was significantly altered. Taken together, these results suggest that sphingolipids containing C26 VLCFAs act as regulatory lipids in the homotypic vacuolar fusion cascade by assembling membrane microdomains that promote the protein and lipid machinery required for the tethering and docking of vacuoles.

Automated quantification of vacuole fusion and lipophagy in Saccharomyces cerevisiae from fluorescence and cryo-soft X-ray microscopy data using deep learning

“…This effect was most pronounced in wild-type cells, where the contribution of multi-vesicular vacuoles went from 25.34% in the absence to more than 60% for the uncorrected data ( Figure 2A ) and 80% for the corrected data ( Figure 2B ) in the presence of myriocin. This result confirms earlier observations, which showed that sphingolipids and very long chain bases are essential for vacuole fusion [ 17 ]. Interestingly, myriocin had a slightly milder effect in both ncr1Δ and npc2∆ cells ( Figure 2 ).…”

“…This could be the direct consequence of impaired delivery of ergosterol into the vacuole membrane in the absence of the Ncr1-Npc2 transport system, in line with our previous results, and the absolute requirement of ergosterol for vacuole fusion [ 8 , 43 , 44 ]. Alternatively, it could be the consequence of other factors, such as a disturbed sphingolipid homeostasis, altered TORC signaling or changed iron availability, which all have been observed in ncr1Δ cells [ 16 , 17 , 45 , 46 ]. To explore such possibilities, we have used inhibitors of the aforementioned processes and assessed their impact on vacuole fusion status in wild-type cells and in cells lacking either Ncr1 or Npc2 under starvation conditions.…”

“…This allows for counting the number of LDs inside vacuoles as a measure for lipophagy under various starvation conditions and genetic backgrounds [ 7 , 18 , 69 , 83 , 84 ]. Similarly, vacuole morphology is often assessed by visual inspection, and from the number of attached vesicles the fusion status is inferred [ 11 , 15 , 17 ]. While reliable and largely reproducible between different human observers, visual annotation of vacuole phenotypes is tedious.…”

The very long‐chain fatty acid elongase FgElo2 governs tebuconazole sensitivity and virulence in Fusarium graminearum

“…In Saccharomyces cerevisiae, for example, VLCFAs are associated with the highly curved membrane structures, which appear to be especially important in the endo-lysosomal pathway (Obara et al, 2013;Schneiter et al, 2004). The yeast cells lacking Elo2 or Elo3 display morphological and vacuolar defects (Chung et al, 2003;Hurst et al, 2020). Recently, VLCFAs have been reported to be critical for mating (Villasmil et al, 2016(Villasmil et al, , 2017, osmotic tolerance (Zhu et al, 2020), cell survival and oleic acid-mediated cytotoxicity (Wang et al, 2018) in S. cerevisiae.…”