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External and intrinsic mechanical stress restricts pheromone signaling by direct phosphorylation of the Ste5 scaffold by Pkc1

ABSTRACT: Ste5 and Far1 function as prototypical scaffold proteins activating a MAP-kinase cascade and polarizing yeast cells in response to pheromones. Here we show that Pkc1-dependent phosphorylation of conserved serine residues in their RING-H2 domains down-regulates pheromone induced signaling upon mechanical stress. Phosphorylation of Ste5 on serine 185 by Pkc1 interferes with its membrane-association in vivo by preventing binding to the receptor linked Gbg protein as determined by in vitro assays. Quantitative readouts of pheromone signaling show that Pkc1 induces rapid dispersal of Ste5 from mating projections upon mechanically-induced cell wall stress as well as during cell-cell fusion, leading to inhibition of MAPK activity and polarized growth. Cells expressing non-phosphorylatable Ste5 (Ste5S185A) undergo increased lysis during mechanical stress and cell-cell fusion compared to wild-type controls, and this effect is exacerbated by simultaneous expression of non-phosphorylatable Far1 (Far13A). Taken together, these results uncover a cross-talk mechanism explaining how mating cells inhibit pheromone signaling upon extrinsic or intrinsic mechanical stress to orchestrate polarized growth and cell-cell fusion.

INSTRUMENT(S): LTQ FT, LTQ Orbitrap Velos, LTQ

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Wolfgang Reiter

LAB HEAD: Wolfgang Reiter

PROVIDER: PXD004657 | Pride | 2019-06-17

REPOSITORIES: Pride

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			Action	DRS
	Exp10_Ste5HTB_LTQ_Orbitrap_Velos_MS_in_solution_digest_try_Reiter_27012011.raw	Raw
	Exp11_Ste5HTB_LTQ_Orbitrap_Velos_MS_in_solution_digest_TiO2_Reiter_07022011.raw	Raw
	Exp12_Ste5HTB_ste7d_LTQ_Orbitrap_Velos_MS_in_solution_digest_try_Reiter_27012011.raw	Raw
	Exp13_Ste5HTB_ste7d_LTQ_Orbitrap_Velos_MS_in_solution_digest_TiO2_Reiter_07022011.raw	Raw
	Exp14_Ste5HTB_LTQ_Orbitrap_Velos_MS_in_solution_digest_try_Reiter_25032011.raw	Raw

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Project description:Candida albicans can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of C. albicans and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced - under a specialized set of conditions - to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such "pheromone-stimulated" biofilms with that of "conventional" C. albicans biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as C. albicans Cph1, ortholog of Saccharomyces cerevisiae Ste12. Cph1 regulates mating in C. albicans opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 206 genes that are induced by pheromone signaling during biofilm formation. One of these genes, HGC1, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in C. albicans, and demonstrate that Cph1 is required for the latter, but not the former. 4 condition experiment: white and opaque cells in planktonic and pheromone-induced biofilm conditions with and without alpha pheromone. WT strain (P37005), the tec1 mutant strain and the cph1 mutant strain

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External and intrinsic mechanical stress restricts pheromone signaling by direct phosphorylation of the Ste5 scaffold by Pkc1

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