Dominant suppressor mutation bypasses the sphingolipid requirement for growth of Saccharomyces cells at low pH: role of the CWP2 gene

Abstract

Strains of Saccharomyces cerevisiae termed sphingolipid compensatory (SLC) do not grow at low pH when the cells lack sphingolipids. To begin to understand why sphingolipids are required for growth at low pH, we isolated derivatives of SLC strains, termed low pH resistant (LprR), carrying the LPR suppressor gene that allows growth at pH 4.1 when cells lack sphingolipids. Suppression is due to mutation of a single nuclear gene. The LPR suppressor gene functions, at least in part, by enhancing the ability of cells lacking sphingolipids to generate a net efflux of protons in suspension fluid with a pH range of 4.0-6.0. The LPR suppressor gene also enables cells lacking sphingolipids to maintain their intracellular pH near neutrality when the pH of the suspension fluid is low, unlike cells lacking the suppressor gene, which cannot maintain their intracellular pH in the face of a low external pH. These results demonstrate that some functions(s) of sphingolipids necessary for growth at low pH can be bypassed by a suppressor mutation. Attempts to clone the LPR suppressor gene were not successful, but they led to the isolation of the CWP2 gene, which encodes a major mannoprotein component of the outer cell wall. It was isolated because an increased copy number has the unusual property of increasing the frequency at which LprR strains arise. As we show here, part of the reason for this effect is that the CWP2 gene is essential for generating a net efflux of protons and for controlling intracellular pH in LprR strains that lack sphingolipids. These results suggest new cellular functions for the Cwp2 protein.

Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

“…The Cwp2 protein has a GPI‐anchor added to its carboxy terminus in the endoplasmic reticulum that is removed when the protein is transported to the plasma membrane and attached to the (1→6)‐β‐D‐glucans of the cell wall (van der Vaart et al ., 1995; Kapteyn et al ., 1999b). This major protein plays a role in stabilizing the cell wall and is involved in low‐pH resistance (Skrzypek et al ., 2000). LAS21 (YJL062W) participates in GPI synthesis by adding an ethanolamine phosphate to the (1→6)‐β‐D‐linked mannose of the GPI mannose core (Benachour et al ., 1999).…”