Yeast osmoregulation - glycerol still in pole position

Abstract

In response to osmotic dehydration cells sense, signal, alter gene expression, and metabolically counterbalance osmotic differences. The main compatible solute/osmolyte that accumulates in yeast cells is glycerol, which is produced from the glycolytic intermediate dihydroxyacetone phosphate. This review covers recent advancements in understanding mechanisms involved in sensing, signaling, cell-cycle delays, transcriptional responses as well as post-translational modifications on key proteins in osmoregulation. The protein kinase Hog1 is a key-player in many of these events, however, there is also a growing body of evidence for important Hog1-independent mechanisms playing vital roles. Several missing links in our understanding of osmoregulation will be discussed and future avenues for research proposed. The review highlights that this rather simple experimental system-salt/sorbitol and yeast-has developed into an enormously potent model system unravelling important fundamental aspects in biology.

Keywords: cell cycle delay; gene expression; osmo-sensing; osmo-signaling; osmoregulation; yeast.

Figure 1.

Glycerol metabolism in yeast. Glycerol is produced from the glycolytic intermediate dihydroxyacetone phosphate DHAP. The formation of glycerol is catalyzed by the NAD-dependent glycerol-3-phosphate dehydrogenase, Gpd1 and Gpd2, and glycerol-3-phosphatase, Gpp1 and Gpp2. In addition, there is an alternative pathway for glycerol production via dihydroxyacetone and the enzymes Dak1/Dak2 and Gcy1/Ypr1, however, this pathway does not seem to play a role in glycerol production under osmostress in yeast but have been reported important in this process in the fungi Aspergillus nidulans (de vries 2003). Intracellular accumulation of glycerol is regulated by the aquaglyceroporin Fps1. Glycerol is assimilated by active uptake via the glycerol/H⁺-symporter Stl1, phosphorylated by the glycerol kinase Gut1 and oxidised by the FAD-dependent glycerol-3-phosphate dehydrogenase Gut2 in the mitochondrial membrane to DHAP, which then enters glycolysis/gluconeogenesis. This figure is from (Hohmann 2015) and is published with permission from Springer Nature.

Figure 2.

A) The model of osmo-sensing and osmo-signaling of the HOG pathway in S. cerevisiae. For the Sln1 branch, osmotic stress releases Sln1-dependent inhibition of Ssk2/22 to activate the pathway. For the Sho1 branch, activation requires the membrane-embedded mucin proteoglycans Msb2 or Hkr1 to interact with Sho1 and Ste20 in a complex with the MAPK components. Opy2 is a transmembrane glycoprotein that serves as an anchor for Ste50. Cdc24 and Cdc42 are the cytosolic guanosine triphosphatase (GTPase) and guanine nucleotide exchange factor (GEF) that activate Ste20.This figure is from (Brewster and Gustin 2014) and is published with permission from the American Association for the Advancement of Science. B) Schematic model of the transmembrane interactions between a Sho1 dimer and two Opy2 proteins. Transmembrane (TM) domains of Sho1 and Opy2 are indicated as light purple/blue and red cylinders, respectively. This figure is from (Takayama et al. 2019).

Figure 3.

Saccharomyces cerevisiae has two independent sensing and signaling systems to rapidly increase intracellular glycerol. The figure depicts a schematic representation of the involved components for each system. These processes act synergistically to elevate the intracellular glycerol concentration to counterbalance the external osmotic stress. (A) HOG pathway-mediated control of intracellular glycerol. In unstressed conditions (upper panel) Hog1 is inactive and glycerol is generated as a minor side product of glycolysis to regulate the cytoplasmic NADH/NAD⁺ imbalance during fermentative growth. Produced glycerol escapes the cell through the Fps1 channel that is maintained in its open state by the bound regulators Rgc1 and Rgc2. Upon hyperosmotic stress (bottom panel) components coupled to the Sho1 and Sln1 osmosensors lead to Hog1 phosphorylation and activation. Activated Hog1 increases intracellular glycerol via both phosphorylation of Pkf26 in the cytosol (leading to enhanced glycolytic flux) and phosphorylation of Rgc1 and Rgc2 preventing glycerol efflux through Fps1. Activated Hog1 also enters the nucleus (not depicted in the figure) where it transcriptionally upregulates GPD1 and several other osmostress genes. (B) TORC2-Ypk-mediated control of intracellular glycerol. In unstressed conditions (upper panel) active TORC2-Ypk1 keeps intracellular glycerol level low by enzymatic inhibition via phosphorylation of Gpd1 and by phosphorylation stimulating the open state of Fps1. Upon hyperosmotic stress (bottom panel) the TORC2-dependent phosphorylation of Ypk1 is rapidly down-regulated that leads to that the inhibition of Gpd1 is alleviated, thereby increasing glycerol production. Concomitantly, loss of Ypk1-mediated phosphorylation closes the Fps1 channel, promoting glycerol accumulation. This figure is from (Muir et al. 2015).

Figure 4.

Model over the structure of the transcriptional network activated by Hog1 during KCl-induced osmotic stress. The model depicts that the incoming osmo-signal to Hog1 (thick black arrow) is spread out via Hog1 (medium-sized black arrows) to multiple transcription factors, Sko1, Hot1 and Msn2/4, that cooperated in different ways at different promoters (thin arrows in different colours). The general stress signal to the general transcription factor Msn2/4 (thick blue arrow) and the Hog-dependent signaling via the transcription factors Sko1 and Hot1 are integrated at a subset of the general stress–responsive genes. Targeted genes are grouped denoted by a box, where some genes are indicated by name and the numbers in parenthesis indicate the total number of genes in that group, on the basis of expression and common regulatory mechanisms. Groups are shown only if two or more genes have the same connections between the indicated transcription factors as determined by expression and confirmed by ChIP. Broken lines indicate interactions that exist for only some genes of a group. ‘Prestress’ indicates the regulatory mechanisms during no osmostress conditions (Sko1 repression), while ‘stress’ indicates the transcriptional mechanisms in operation during osmostress. This figure is from (Capaldi et al. 2008) and is published with permission from Springer Nature.

Figure 5.

‘Synthetic’ osmoadaptation in hog1∆ cells where expression of Hog1-dependent osmostress-induced genes (GPD1 and GPP2) was rewired under the control of a Fus3/Kss1-dependent FUS1 promoter. (A) Experimental design for synthetic osmoadaptation in hog1∆ using crosstalk for the production of the key-enzymes Gpd1 and Gpp2 in the glycerol biosynthesis pathway. (B) Synthetic induction of GPD1 and GPP2 strongly suppresses osmosensitivity in YPD + 0.8 M NaCl medium of the hog1∆ strain. GPD1^4A stands for the unphosphorylated form of Gpd1 (four serines in the N-terminus converted into alanine) with a constitutively high enzymatic activity. (C) The intracellular accumulation of glycerol in the strains in B. This figure is from (Babazadeh et al. 2014).