Rgc2 Regulator of Glycerol Channel Fps1 Functions as a Homo- and Heterodimer with Rgc1

The plasma membrane aquaglyceroporin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Fps1 functions as a homotetramer, and control of its channel activity in response to hyperosmotic shock involves a redundant pair of fungus-specific regulators, Rgc1 and Rgc2 (regulators of the glycerol channel), and the mitogen-activatd protein kinase (MAPK) Hog1 (high-osmolarity glycerol response). Rgc1 and Rgc2 maintain Fps1 in an open-channel state by binding to its C-terminal cytoplasmic domain. Phosphorylation of Rgc1 and Rgc2 by Hog1 induces their eviction from Fps1 and consequent channel closure. In the absence of Fps1 channel function, cells experience chronic cell wall stress, which may be exploited for antifungal drug development. We show here that Rgc1 and Rgc2 form homodimers and heterodimers with each other and that dimer formation of Rgc2 is mediated by its N-terminal domain. Mutations that prevent Rgc2 dimerization block its ability to open Fps1. Therefore, the Rgc-Rgc dimer interface might be an attractive drug target.     

Identification of Positive Regulators of the Yeast Fps1 Glycerol Channel

The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.     

Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1

Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Delta and pbs2Delta cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Delta cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.     

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.     

Hog1p mitogen-activated protein kinase determines acetic acid resistance in Saccharomyces cerevisiae

When glucose-repressed, Saccharomyces cerevisiae cannot use acetic acid as a carbon source and is inhibited in growth by high levels of this compound, especially at low pH. Cultures exposed to a 100 mM acetate stress activate both the Hog1p and Slt2p stress-activated MAP kinases. Nevertheless, only active Hog1p, not Slt2p, is needed for the acquisition of acetate resistance. Hog1p undergoes more rapid activation by acetate in pH 4.5, than in pH 6.8 cultures, an indication that the acid may have to enter the cells in order to generate the Hog1p activatory signal. Acetate activation of Hog1p is absent in the ssk1Delta and pbs2Delta mutants, but is present in sho1Delta and ste11Delta, showing that it involves the Sln1p branch of the high-osmolarity glycerol (HOG) pathway signaling to Pbs2p. In low-pH (pH 4.5) cultures, the acetate-activated Hog1p, although conferring acetate resistance, does not generate the GPD1 gene or intracellular glycerol inductions that are hallmarks of activation of the HOG pathway by hyperosmotic stress.     

Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological activity

Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity.     

Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.     

A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p

The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydstr?m, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 6337-6345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1Delta strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane.     

A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans

The Hog1 mitogen-activated protein kinase (MAPK) plays a central role in stress responses in the human pathogen Candida albicans. Here, we have investigated the MAPK kinase kinase (MAPKKK)-dependent regulation of the pathway. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, which is regulated by three MAPKKKs (Ssk2, Ssk22, and Ste11), our results demonstrate that Hog1 in C. albicans is regulated by a single MAPKKK Ssk2. Deletion of SSK2 results in comparable stress and morphological phenotypes exhibited by hog1Delta cells, and Ssk2 is required for the stress-induced phosphorylation and nuclear accumulation of Hog1, and for Hog1-dependent gene expression. Furthermore, phenotypes associated with deletion of SSK2 can be circumvented by expression of a phosphomimetic mutant of the MAPKK Pbs2, indicating that Ssk2 regulates Hog1 via activation of Pbs2. In S. cerevisiae, the Hog1 pathway is also regulated by the MAPKKK Ste11. However, we can find no connection between Ste11 and the regulation of Hog1 in C. albicans. Furthermore, expression of a chimeric Pbs2 protein containing the Ste11-dependent regulatory region of S. cerevisiae Pbs2, fails to stimulate Ste11-dependent stress signaling in C. albicans. Collectively, our data show that Ssk2 is the sole MAPKKK to relay stress signals to Hog1 in C. albicans and that the MAPK signaling network in C. albicans has diverged significantly from the corresponding network in S. cerevisiae.     

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen.

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.     

Yeast aquaglyceroporins use the transmembrane core to restrict glycerol transport

Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.     

Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1

The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Delta strain due to hyperactivation of the HOG pathway. In this work, we show that PTC2 also suppresses sln1Delta lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, approximately 12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to approximately 3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.     

MAPKKK-independent regulation of the Hog1 stress-activated protein kinase in Candida albicans

The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2(AA)). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2(DD)) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2(DD) cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2(DD) cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2(AA) and Pbs2(DD) cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2(AA) cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.