Ste20 and Cla4 modulate the expression of the glycerol biosynthesis enzyme Gpd1 by a novel MAPK-independent pathway

Ifeoluwapo Matthew Joshua,Thomas Höfken

doi:10.1016/j.bbrc.2019.07.072

Ifeoluwapo Matthew Joshua, Thomas Höfken

Open Access

https://doi.org/10.1016/j.bbrc.2019.07.072

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Abstract

p21-activated kinases (PAKs) are important signalling molecules with a wide range of functions. In budding yeast, the main PAKs Ste20 and Cla4 regulate the response to hyperosmotic stress, which is an excellent model for the adaptation to changing environmental conditions. In this pathway, the only known function of Ste20 and Cla4 is the activation of a mitogen-activated protein kinase (MAPK) cascade through Ste11. This eventually leads to increased transcription of glycerol biosynthesis genes, the most important response to hyperosmotic shock. Here, we show that Ste20 and Cla4 not only stimulate transcription, they also bind to the glycerol biosynthesis enzymes Gpd1, Gpp1 and Gpp2. Protein levels of Gpd1, the enzyme that catalyzes the rate limiting step in glycerol synthesis, positively correlate with glucose availability. Using a chemical genetics approach, we find that simultaneous inactivation of STE20 and CLA4 reduces the glucose-induced increase of Gpd1 levels, whereas the deletion of either STE20 or CLA4 alone has no effect. This is also observed for the hyperosmotic stress-induced increase of Gpd1 levels. Importantly, under both conditions the deletion of STE11 has no effect on Gpd1 induction. These observations suggest that Ste20 and Cla4 not only have a role in the transcriptional regulation of GPD1 through Ste11. They also seem to modulate GPD1 expression at another level such as translation or protein degradation.

Highlights

The p21-activated kinases (PAKs) are highly conserved effectors of the Rho GTPases Cdc42 and Rac
Out of these only GPD1 is essential for osmoadaptation, and the upregulation of GPD1 and GPP2 is sufficient for efficient adaptation to hyperosmotic stress, highlighting the central role that glycerol biosynthesis plays in osmoadaptation [12]
Suggest that Ste20 and Cla4 might have a function associated with Gpd1 such as aerobic glycerol biosynthesis, in particular in response to hyperosmotic stress, rather than a process associated with Gpd2 such as anaerobic glycerol synthesis

Summary

Introduction

The p21-activated kinases (PAKs) are highly conserved effectors of the Rho GTPases Cdc and Rac. Ste activates three different mitogen-activated protein kinase (MAPK) cascades regulating the hyperosmotic stress response, filamentation and mating [1,4e7]. These pathways are excellent models for the adaptation to changing environmental conditions. The two homologous NADH-dependent glycerol 3-phosphate dehydrogenases Gpd and Gpd convert dihydroxyacetone phosphate to glycerol 3phosphate [13e15], and the two homologous glycerol 3phosphatases Gpp and Gpp metabolize glycerol 3-phosphate to glycerol [16] Despite their similarities the homologous enzymes only have partially overlapping functions. Hyperosmotic stress leads to the Hog1-dependent upregulation of expression of GPD1, GPP2 and hundreds of other genes [10,11,14,16] Out of these only GPD1 is essential for osmoadaptation, and the upregulation of GPD1 and GPP2 is sufficient for efficient adaptation to hyperosmotic stress, highlighting the central role that glycerol biosynthesis plays in osmoadaptation [12]. We examined further links between glycerol biosynthetic enzymes and the PAKs Ste and Cla

Yeast strains and plasmids

Growth conditions and inhibitor treatment

Split-ubiquitin technique

Cell extracts and immunoblotting

Ste20 and Cla4 bind to glycerol biosynthesis enzymes

Gpd1 expression and hyperosmotic stress response