Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling

Abstract

The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate 'membrane compartment occupied by Wsc1' (MCW) shows moderate overlap with other Wsc-type sensors, but not with those of the Mid-type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine-rich head group near the N-terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis.

Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion

“…For Western blot analyses, crude extracts prepared as above were mixed with SDS sample buffer (final concentrations: 60 mM Tris/HCl, pH 6.8, 10% glycerol, 2% SDS, 0.005% Bromphenol Blue) and heated to 98 • C for 5 min, prior to separation on precast 10% SDS-polyacrylamide gels (LTF-Labortechnik GmbH and Co KG Wasserburg, Germany). Blotting and preparation for immunological detection was done as explained in [54]: after completion of electrophoresis, gels were transferred to a nitrocellulose membrane (Whatman GmbH, Dassel) with the "Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell" (Bio-Rad). Membranes were blocked for 1 to 3 h at room temperature with TBST (20 mM Tris, 150 mM NaCl, pH adjusted to 7.6 with HCl, 0.05% Tween 20) containing 3% bovine serum albumin (BSA, Roth, Karlsruhe, Germany).…”

Glycolytic Functions Are Conserved in the Genome of the Wine Yeast Hanseniaspora uvarum, and Pyruvate Kinase Limits Its Capacity for Alcoholic Fermentation

“…This concentration was also used for strains carrying endogenous ScPFK genes. Secondary anti-rabbit antibodies coupled to an infrared dye with fluorescence at 700 nm were then employed for the detection of PFK signals using the Odyssey imaging device as described previously (91). Contrast, brightness, and frames of the images were adjusted by using Corel PhotoPaint, which was applied only to the entire images shown in the figures and not to single bands.…”

Lateral plasma membrane compartmentalization links protein function and turnover

“…The network-like membrane compartment occupied by Pma1 (MCP), on the other hand, is enriched in the H + -ATPase Pma1, which is essential for pH homeostasis and maintenance of the PM potential. Additional domains have been proposed that contain the sterol transporters Lct3/4 (Murley et al, 2017), the sensor of cell-wall integrity Wsc1 (Kock et al, 2016) or the target of rapamycin kinase complex 2 (TORC2; Berchtold & Walther, 2009). TORC2 has a wide range of targets and regulates actin polymerization, endocytosis, and sphingolipid synthesis (Gaubitz et al, 2016).…”