A glutamine sensor that directly activates TORC1.

TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells.

Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K.

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae.

To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.

Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha.

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase.

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1(+) cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1.

Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway.

To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor.

Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1.

The yeast high osmolarity glycerol (HOG) pathway activates the Hog1 MAP kinase, which coordinates adaptation to high osmolarity conditions. Here we demonstrate that the four-transmembrane (TM) domain protein Sho1 is an osmosensor in the HKR1 sub-branch of the HOG pathway. Crosslinking studies indicate that Sho1 forms planar oligomers of the dimers-of-trimers architecture by dimerizing at the TM1/TM4 interface and trimerizing at the TM2/TM3 interface. High external osmolarity induces structural changes in the Sho1 TM domains and Sho1 binding to the cytoplasmic adaptor protein Ste50, which leads to Hog1 activation. Besides its osmosensing function, the Sho1 oligomer serves as a scaffold. By binding to the TM proteins Opy2 and Hkr1 at the TM1/TM4 and TM2/TM3 interface, respectively, Sho1 forms a multi-component signalling complex that is essential for Hog1 activation. Our results illuminate how the four TM domains of Sho1 dictate the oligomer structure as well as its osmosensing and scaffolding functions.

Yeast osmosensors Hkr1 and Msb2 activate the Hog1 MAPK cascade by different mechanisms.

To cope with environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the mitogen-activated protein kinase (MAPK) Hog1, which controls an array of osmoadaptive responses. Two independent, but functionally redundant, osmosensing systems involving the transmembrane sensor histidine kinase Sln1 or the tetraspanning membrane protein Sho1 stimulate the Hog1 MAPK cascade. Furthermore, the Sho1 signaling branch itself also involves the two functionally redundant osmosensors Hkr1 and Msb2. However, any single osmosensor (Sln1, Hkr1, or Msb2) is sufficient for osmoadaptation. We found that the signaling mechanism by which Hkr1 or Msb2 stimulated the Hog1 cascade was specific to each osmosensor. Specifically, activation of Hog1 by Msb2 required the scaffold protein Bem1 and the actin cytoskeleton. Bem1 bound to the cytoplasmic domain of Msb2 and thus recruited the kinases Ste20 and Cla4 to the membrane, where either of them can activate the kinase Ste11. The cytoplasmic domain of Hkr1 also contributed to the activation of Ste11 by Ste20, but through a mechanism that involved neither Bem1 nor the actin cytoskeleton. Furthermore, we found a PXXP motif in Ste20 that specifically bound to the Sho1 SH3 (Src homology 3) domain. This interaction between Ste20 and Sho1 contributed to the activation of Hog1 by Hkr1, but not by Msb2. These differences between Hkr1 and Msb2 may enable differential regulation of these two proteins and provide a mechanism through Msb2 to connect regulation of the cytoskeleton with the response to osmotic stress.

Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor.

Membrane localization of the Ste11 MAPKKK is essential for activation of both the filamentous growth/invasive growth (FG/IG) MAP kinase (MAPK) pathway and the SHO1 branch of the osmoregulatory HOG MAPK pathway, and is mediated by binding of the Ste50 scaffold protein to the Opy2 membrane anchor. We found that Opy2 has two major (CR-A and CR-B), and one minor (CR-D), binding sites for Ste50. CR-A binds Ste50 constitutively and can transmit signals to both the Hog1 and Fus3/Kss1 MAPKs. CR-B, in contrast, binds Ste50 only when Opy2 is phosphorylated by Yck1/Yck2 under glucose-rich conditions and transmits the signal preferentially to the Hog1 MAPK. Ste50 phosphorylation by activated Hog1/Fus3/Kss1 MAPKs downregulates the HOG MAPK pathway by dissociating Ste50 from Opy2. Furthermore, Ste50 phosphorylation, together with MAPK-specific protein phosphatases, reduces the basal activity of the HOG and the mating MAPK pathways. Thus, dynamic regulation of Ste50-Opy2 interaction fine-tunes the MAPK signaling network.

Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK.

The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG-specific Kss1 MAPK is activated by a combination of an O-glycosylation defect caused by disruption of the gene encoding the protein O-mannosyltransferase Pmt4, and an N-glycosylation defect induced by tunicamycin. The O-glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG-specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.

Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway.

To cope with life-threatening high osmolarity, yeast activates the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1-independent manner.

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.

Salt stress induces the expression of Schizosaccharomyces pombe och1+, which encodes an initiation-specific alpha-1,6-mannosyltransferase for N-linked outer chain synthesis of cell wall mannoproteins.

The Schizosaccharomyces pombe Och1p is required for the initiation of outer chain elongation of N-linked oligosaccharides. In this report, we investigated the transcriptional control of the S. pombe och1+ gene and found that the expression of the och1+ gene was not regulated during the cell cycle, but was induced by NaCl and KCl through a transcription factor, Atf1p.

Mutation of TRS130, which encodes a component of the TRAPP II complex, activates transcription of OCH1 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Och1p is required for the initiation of the mannose outer chain elongation of cell wall mannoproteins. A trs130(ts1) mutant that showed an elevated expression of the OCH1-reporters was isolated in our laboratory. In this study, we attempted to gain an understanding of the relationship between the defect in the Trs130p function and the elevated expression of OCH1. The trs130(ts1) mutant showed calcofluor white-resistant and hygromycin B-sensitive phenotypes, indicating that the mutant is defective in cell wall integrity. YPT31 and YPT32, the multi-copy suppressors of the trs130(ts1) mutant, differed in the extent of suppression of the mutant phenotypes and in their genetic interaction with TRS130. Our results suggest that the blocking of vesicle transport by a loss of the Trs130p function causes some defect in the cell wall mannoproteins, which leads to the elevated expression of OCH1 through the Skn7p function, to compensate for the defect.