TRS85 and LEM3 suppressor mutations rescue stress hypersensitivities caused by lack of structural diversity of complex sphingolipids in budding yeast.

The budding yeast Saccharomyces cerevisiae can synthesise 15 subtypes of complex sphingolipids, and this structural diversity is thought to be the molecular basis that enables the range of biological functions of complex sphingolipids. Through analyses of yeast mutants with various deletion combinations of complex-sphingolipid-metabolising enzyme genes (CSG1, CSH1, IPT1, SUR2 and SCS7), it was previously shown that less structural diversity of complex sphingolipids leads to increased sensitivity to multiple environmental stresses, with impaired plasma-membrane and cell-wall integrity. In this study, we screened for suppressor mutations that can alleviate the stress hypersensitivities of csg1Δ csh1Δ sur2Δ scs7Δ (ccssΔ) cells. Mutations of trafficking protein particle complex III-specific subunit 85 (TRS85; encodes a component of the TRAPPIII complex, involved in membrane trafficking) and phospholipid-transporting ATPase Dnf2 (DNF2; encodes the plasma-membrane glycerophospholipid flippase) were identified as suppressor mutations. Loss of Trs85 or phospholipid-transporting ATPase accessory subunit Lem3 (LEM3; encodes a regulatory subunit of Dnf2) differed in the type of stress being conferred resistance to ccss∆ cells. Furthermore, it was also found that impaired plasma-membrane and cell-wall integrities in ccssΔ cells were suppressed by trs85∆ but not lem3∆. Moreover, ccss∆ cells exhibited abnormal localisation of yeGFP-Snc1 in endosomes, which is suppressed by trs85∆ but not lem3∆. Overexpression of GTP-binding protein Ypt1, which is regulated by TRAPPIII and involved in vesicular trafficking, exacerbated plasma-membrane integrity abnormalities and stress sensitivities in ccss∆ cells. Thus, it was suggested that TRS85 and LEM3 deletion confer stress tolerances to ccssΔ cells through distinct mechanisms. These findings will provide insights into the physiological significance of the structural diversity of complex sphingolipids.