Characterization of the Wsc1 Protein, a Putative Receptor in the Stress Response of Saccharomyces cerevisiae

Abstract

Wsc1p, Wsc2p, Wsc3p, and Wsc4p, members of a novel protein family in the yeast Saccharomyces cerevisiae, are putative sensors or receptors in the stress response. Genetic characterization suggests that the WSC family are upstream regulators of the stress-activated PKC1-MAP kinase cascade and are required for the heat shock response and for maintenance of cell wall integrity. The Wsc proteins share sequence characteristics: at their N terminus they have a cysteine motif and a serine/threonine-rich domain predicted to be extracellular, a hydrophobic domain suggested to be transmembranous, and a variable, highly charged C terminus that may be involved in intracellular signaling. Although a role for the WSC genes in maintenance of cell wall integrity has been firmly established, little is known about the properties of the proteins. As reported here, to study its properties in vivo, we epitope tagged the Wsc1 protein. Wsc1p was found to fractionate with the membrane pellet after high-speed centrifugation. Extraction experiments show that Wsc1p is an integral membrane protein present in two forms: one solubilized by detergent, the other Triton X-100 insoluble. Our results also show that Wsc1p is glycosylated and phosphorylated. To characterize the contribution of different domains to the function of Wsc1p, we generated various deletion constructs. Analysis of the properties and function of the mutant proteins shows that the predicted extracellular serine/threonine-rich domain is required for Wsc1p functionality, as well as its glycosylation. A mutant Wsc1 protein lacking the putative transmembrane domain is not functional and partitions to the soluble fraction. Overexpression of full-length Wsc1p inhibits cell growth, with the N terminus alone being sufficient for this inhibition. This suggests that Wsc1p may function in a complex with at least one other protein important for normal cell growth.

Cells under pressure: how yeast cells respond to mechanical forces

“…Wsc1 is among the best described mechano-sensors and coordinates mechano-transduction and cell wall integrity in budding yeast [48,[69][70][71]. This transmembrane protein accumulates predominantly at sites of polarized growth and functions as an upstream regulator of the Cell Wall Integrity pathway (CWI) that adjusts cell wall synthesis in response to chemical or heat stress [71][72][73]. Indeed, wsc1 cells are sensitive to cell wall damaging agents [72] and often lyse during polarized growth in processes such as budding and shmooing.…”

Regulation of Peroxisome Homeostasis by Post-Translational Modification in the Methylotrophic Yeast Komagataella phaffii

“…Our previous studies revealed that WSC family proteins act as methanol-sensing machinery for regulating methanol-induced gene expression in K. phaffii ( Ohsawa et al, 2017 ) . WSC family proteins were identified as “nanospring” mechanosensors of cell wall damage caused by high temperatures and hypo-osmotic stress in Saccharomyces cerevisiae (ScWsc1/ScSlg1) ( Lodder et al, 1999 ; Dupres et al, 2009 ). In the K. phaffii genome , three WSC family protein-encoding genes have been identified ( KpWSC1, KpWSC2 , and KpWSC3 ).…”

Is there anyone out there?—Single-molecule atomic force microscopy meets yeast genetics to study sensor functions

“…24 Deletions of WSC2, WSC3, and MTL1 lack strong phenotypes under most laboratory growth conditions and only slightly aggrevate those of a wsc1 and/or mid2 deletion in multiple combinations. 20,25 Therefore, the encoded sensors are thought to be less important and most studies have been directed towards Wsc1 sensor function.…”

“…each of these regions is indispensable for activation of the CWI pathway upon extracellular stress. 20,25 The CRD displays significant similarity to a sequence from a fungal exoglucanase, which led to the hypothesis that it may interact with cell wall glucans. 9 Moreover, mutants with defects in O-mannosylation (pmt1 pmt3), which would cause a severe under-glycosylation of the STR sequences of all CWI sensors, also show cell lysis defects similar to wsc1 mid2 double deletions, which again are osmo-remedial.…”