Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

Abstract

We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2delta and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2delta) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Keywords Yeast middle dot SLT2 middle dot MAP-kinase middle dot Caffeine

Regulatory Mechanisms for Modulation of Signaling through the Cell Integrity Slt2-mediated Pathway in Saccharomyces cerevisiae

“…This is consistent with the observation that mutants affected in the Slt2 pathway display a vanadate and caffeine-sensitive growth phenotype that is remedied by osmotic stabilization of the cells (20,42,43). The fact that the presence of 1 M sorbitol also prevents induced activation of the cell integrity pathway by both agents suggests that these might alter the cell surface.…”

“…Pathway-The ability of sorbitol to rescue the sensitivity of mutants affected in the cell integrity pathway to caffeine (42) and vanadate (data not shown) and to prevent Slt2 phosphorylation induced by these agents suggests that these compounds could be perturbing cell wall stability. In this case, activation of the pathway in response to these stimuli would be mediated by the previously identified cell surface sensors.…”

“…Because sensitivity to caffeine and vanadate is a characteristic phenotype in mutants affected in the Pkc1 pathway (20,42,43), it was tempting to speculate that activation of this pathway is essential for resistance to these compounds. Therefore, we analyzed the effect of caffeine and vanadate on Slt2 phosphorylation and compared it with that of thermal stress.…”

“…In order to test whether Slt2 F54 behavior was a particular feature of this mutant protein or due to the TD28 background, we repeated similar experiments with the DL443 strain, bearing a different inactive allele, namely slt2D35, in another genetic background (42). However, since this strain displays a strong lytic phenotype at 39°C, the exposure time was lowered to 1 h in order to avoid high loss of viability.…”

Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases

“…The Mpk1 cascade is responsible for the integrity of the cell wall of the yeast (58 -60). Therefore, exposure of yeast cells to drugs such as caffeine or to stimuli that damage the cell wall induces this pathway (61). Cells that are defective in components of the pathway, such as those lacking the MAPKKK Bck1, the redundant MAPKKs, Mkk1 and Mkk2, or the Mpk1 itself, cannot grow under these conditions.…”