Temperature-Induced Expression of Yeast FKS2 Is under the Dual Control of Protein Kinase C and Calcineurin

Abstract

FKS1 and FKS2 are alternative subunits of the glucan synthase complex, which is responsible for synthesizing 1,3-␤-glucan chains, the major structural polymer of the Saccharomyces cerevisiae cell wall. Expression of FKS1 predominates during growth under optimal conditions. In contrast, FKS2 expression is induced by mating pheromone, high extracellular [Ca 2؉ ], growth on poor carbon sources, or in an fks1 mutant. Induction of FKS2 expression in response to pheromone, CaCl 2 , or loss of FKS1 function requires the Ca 2؉ /calmodulindependent protein phosphatase calcineurin. Therefore, a double mutant in calcineurin (CNB1) and FKS1 is inviable due to a deficiency in FKS2 expression. To identify novel regulators of FKS2 expression, we isolated genes whose overexpression obviates the calcineurin requirement for viability of an fks1 mutant. Two components of the cell integrity signaling pathway controlled by the RHO1 G protein (MKK1 and RLM1) were identified through this screen. This signaling pathway is activated during growth at moderately high temperatures. We demonstrate that calcineurin and the cell integrity pathway function in parallel, through separable promoter elements, to induce FKS2 expression during growth at 39°C. Because RHO1 also serves as a regulatory subunit of the glucan synthase, our results define a regulatory circuit through which RHO1 controls both the activity of this enzyme complex and the expression of at least one of its components. We show also that FKS2 induction during growth on poor carbon sources is a response to glucose depletion and is under the control of the SNF1 protein kinase and the MIG1 transcriptional repressor. Finally, we show that FKS2 expression is induced as cells enter stationary phase through a SNF1-, calcineurin-, and cell integrity signalingindependent pathway.The cell wall of the budding yeast Saccharomyces cerevisiae is required to maintain cell shape and integrity (4,20). Vegetative proliferation requires that the cell remodels its wall to accommodate growth. The main structural components responsible for the rigidity of the yeast cell wall are 1,3-␤-linked glucan polymers with some branches through 1,6-␤ linkages. The biochemistry of the yeast enzyme complex that catalyzes the synthesis of 1,3-␤-glucan chains has been studied extensively (15, 29), and three genes that encode components of this complex have been identified. A pair of closely related genes, FKS1 and FKS2, encode alternative subunits of the 1,3-␤-glucan synthase (GS) (8,15,28,36). Either FKS1 or FKS2 function is sufficient for GS activity and cell viability. Additionally, the Rho1 GTPase is an essential regulatory subunit of the GS complex, serving to stimulate GS activity in a GTP-dependent manner (9, 35).A second essential function of RHO1 is to regulate the cell integrity signaling pathway by binding and activating protein kinase C (19, 33), which is encoded by PKC1 (25). Loss of PKC1 function results in a cell lysis defect that is attributable to a deficiency in cell wall construction (23,24,34 ...

Reciprocal Regulation between Slt2 MAPK and Isoforms of Msg5 Dual-specificity Protein Phosphatase Modulates the Yeast Cell Integrity Pathway

“…1B shows that the absence of Msg5 leads to an increased sensitivity of cells to Congo Red, a compound that interferes with cell wall assembly (33). Consistent with a role for Msg5 in cell wall construction, we also observed that msg5⌬ cells transformed with a FKS2-lacZ reporter displayed double the ␤-galactosidase activity of the wild type cells (data not shown), reflecting the transcriptional induction of FKS2, a known output of the cell integrity pathway (34). These studies support the notion that Msg5 regulates cell wall physiology, and taken together, all of these findings indicate that this phosphatase is a key regulator of the cell integrity pathway.…”

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

“…Movement of Fks1p driven by actin is required for the construction of a uniform and solid cell wall (Utsugi et al, 2002). Transcription of FKS2 is up-regulated in response to cell wall stress induced by heat, cell wall mutations, and cell wall-perturbing agents (Zhao et al, 1998;de Nobel et al, 2000;Lagorce et al, 2003;Garcia et al, 2004). Deletion of KRE5 leads to a 114-fold up-regulation of FKS2 in response to an impaired cell wall (Kapteyn et al, 1999).…”

Presence of the Fps1p aquaglyceroporin channel is essential for Hog1p activation, but suppresses Slt2(Mpk1)p activation, with acetic acid stress of yeast

“…Cells of the fks1D mutant show an elevated basal level of phospho-Slt2p (Reinoso- Martin et al, 2003) and compensate for their reduced cell wall glucan content by increasing their chitin content as well as the expression of the second glucan synthase gene, FKS2. This has raised the suggestion of a signalling feedback from the cell wall via Wsc1p to monitor the activity of Fks1p (Zhao et al, 1998). Therefore, the Wsc1p-dependent phosphorylation of Slt2p in response to acetate stress (Fig.…”