Metabolic respiration induces AMPK- and Ire1p-dependent activation of the p38-Type HOG MAPK pathway

Abstract

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.

Figure 1. Gene expression profiling by RNA seq analysis and qPCR.

A) Genes induced by salt, tunicamycin (TUN), or galactose (GAL). All RNA seq comparisons are provided in Table S1. B) Genes induced in a Pbs2p-dependent manner under the indicated conditions. Genes outlined by the dark blue circle (Pbs2p-dependent GAL specific) were functionally annotated in a pie chart in Fig. S2. C) Heat map of genes induced by the indicated stresses. Common targets and targets unique to each stimulus is shown. Asterisk, target of ESR. D) qPCR of HOG pathway target mRNAs in wild type and the pbs2Δ mutant grown in glucose (GLU, YEPD) and galactose (GAL, YEP-GAL). Error bars indicate +/−S.E.M. of three independent experiments. Actin (ACT1) mRNA was used as a control. E) Activity of p8XCRE-lacZ in wild-type cells (PC313) and pbs2Δ mutant (PC5035) grown in YEPD (5.5 hr), YEP-GAL (5.5 hr), and YEPD+0.4 M KCl (30 min). F) qPCR of Ste12p target mRNAs in wild type (PC538) and the ste12Δ (PC2382) mutant grown in glucose (YEPD) and galactose (YEP-GAL). See panel D for details.

Figure 2. Comparison of HOG pathway activation by galactose and osmotic stress.

For all phosphoblots involving Hog1p and Kss1p, the sizes of proteins are P∼Hog1p (∼49 kDa), Hog1p (∼49 kDa), P∼Kss1p (∼43 kDa), Kss1p (∼43 kDa), and Pgk1p (∼45 kDa). Pgk1p was used as a loading control. Asterisk (*) refers to a background band detected by the Kss1p antibody. Basal P∼Hog1p and P∼Kss1p showed variable levels under un-inducing conditions. A) Wild type cells (PC538) cells were grown to mid-log phase (∼5.5 hrs) in YEPD (GLU) or YEP-GAL (GAL) media and evaluated by immunoblot analysis for phosphorylation of the MAPKs Hog1p and Kss1p. B) Graph of P∼Hog1p levels under the indicated conditions, as determined by ImageJ analysis. C) Time-course analysis. Wild-type cells (PC538) were grown to mid-log phase and transferred to media containing salt (YEPD+0.4 M KCl) or galactose (YEP-GAL) for the indicated times. D) Extended time course of Hog1∼P during growth in galactose. E) Combinatorial analysis of the response to osmotic stress and galactose. Cells were grown to mid-log phase in YEPD, YEP-GAL, or YEPD+0.4M KCl, which was added to the cells growing in YEPD for 5 min. F) P∼Hog1p levels in cells shifted from galactose (YEP-GAL) to glucose (YEPD) for the indicated time points. Cells in YEP-GAL media were harvested by centrifugation, washed twice in water, and resuspended in YEPD for the indicated time points. G) P∼Hog1p levels during growth in 0.4M KCl and galactose in mutants lacking Ssk1p or Ste11p branches of the HOG pathway. Wild type cells (PC538), and the ssk1Δ (PC1523), ssk2Δ (PC6086), ssk22Δ (PC6085), ssk2Δ ssk22Δ (PC6031), ste11Δ (PC3861), ste11Δ ssk1Δ (PC2061), pbs2Δ (PC2053) and hog1Δ (PC6047) mutants were grown in YEP-GAL medium or YEPD medium containing 0.4M KCl for 5 min.

Figure 3. Role of increased metabolic respiration and Snf1p in activation of the HOG pathway.

A) Immunoblot showing P∼Hog1p levels in cells grown in glucose (YEPD), galactose (YEP-GAL) or glucose and galactose (YEPD+2% GAL). B) Wild-type cells (PC6016) and the gal3Δ, gal4Δ, gal7Δ and gal10Δ mutants grown in YEP-GAL. C) P∼Hog1 levels in cells grown under the indicated conditions for 3 h with or without antimycin, ANT. D) Wild type (PC538) and the aco1Δ (PC3912), fum1Δ (PC6152), mdh1Δ (PC6153) and kgd1Δ (PC6155) and idh1Δ (PC6154) mutants were grown in galactose for 5.5 hrs. E) Wild-type cells (PC538), and the snf1Δ (PC560), mig1Δ (PC4843) and snf1Δ mig1Δ (PC6076) mutants were grown in YEP-GAL medium for 5.5 hrs.

Figure 4. Role of the UPR in mediating HOG pathway activation during growth in galactose and in response to protein glycosylation deficiency.

A) P∼Hog1p and P∼Kss1p levels in the pmi40-101 mutant (PC244) grown in YEPD+/−50 mM MAN (mannose) for 5.5 hrs. B) The pmi40-101 (PC244) and pmi40-101 ire1Δ (PC6044) mutants were grown in YEPD medium +/−50 mM MAN. C) Activity of the UPRE-lacZ reporter under the indicated conditions. H₂O₂ (5 mM; 3 hr), DTT (4 mM; 3 hr), KCl (1M; 30 min), TUN (2.5 µg; 3 hr), galactose (2%, 5.5 hr), MYR (myriocin) (5 µg; 3 hr), SD (+/−nitrogen; 5.5 hr), pmi40-101 (+/− MAN; 5.5 hr), ANT (2.5 µg; 3 hr). D) P∼Hog1p levels in the ire1Δ mutant grown in galactose. Wild-type cells (PC538) and the ire1Δ mutant (PC6048) were grown in YEP-GAL medium.

Figure 5. Cross-inhibition between the filamentous growth and HOG pathways during growth in galactose.

A) Morphology of wild-type cells (PC538) and the pbs2Δ mutant (PC2053), grown on YEPD and YEP-GAL for 24 hrs. Bar, 5 microns. B) pFRE-lacZ reporter activity in wild-type cells (PC313) and the pbs2Δ mutant (PC5035) in YEP-GAL medium. C) Role of protein tyrosine phosphatases in P∼Hog1p activity in galactose. Wild-type cells (PC538), and the ptp2Δ (PC6156), ptp3Δ (PC6157) and ptp2Δ ptp3Δ double mutant (PC6158) were grown in YEPD and YEP-GAL media for 5.5 hrs. D) P∼Kss1p activity in wild-type cells and the pbs2Δ mutant (PC2053) grown in YEP-GAL medium over a time course as indicated. E) P∼Hog1p activity in the kss1Δ mutant (PC620) grown in YEP-GAL medium for the times indicated. F) qPCR showing the relative expression of STE12 mRNA in the wild-type (PC538), pbs2Δ (PC2053) and ste12Δ (PC2382) mutant cells. Error bars indicate +/− standard error mean of three independent experiments. Actin (ACT1) mRNA was used as a control. G) Ste12p-HA protein levels in the wild-type and pbs2Δ strains. Hog1p levels by immunoblot analysis are also shown.

Figure 6. Role of the HOG and filamentous growth pathways in growth in galactose and effect of the inhibitory role of the HOG pathway on filamentous growth pathway outputs.

A) Serial dilutions of wild-type (PC313), ste7Δ (PC4928), pbs2Δ (PC5035) and ste7Δ pbs2Δ (PC6272) cells were spotted on YEPD and YEP-GAL media. B) Morphology of wild-type cells (PC538), the pbs2Δ mutant (PC2053), the ste7Δ mutant (PC4982), and the ste7Δ pbs2Δ double mutant (PC6272) grown YEP-GAL media for 24 hrs. Bar, 5 microns. C) Septin staining of wild-type and pbs2Δ cells harboring the pCdc12p-GFP plasmid. Cells were grown to mid-log phase in YEPD. D) Mat formation in cells lacking the filamentous growth or HOG pathways. Wild-type (PC538), flo11Δ (PC1029), and pbs2Δ (PC2053) strains were grown in YEPD medium for 16 hrs and then spotted onto low agar (0.3%) YEP-GAL medium for 3 d at 30°C. Bar, 1 cm.

Figure 7. MAPK responses in C. albicans during growth in galactose.

A) Immunoblot analysis of P∼CaHog1p. Wild-type (PC6111) cells were grown in YEPD and YEP-GAL medium (5.5 hrs) and treated with TUN (tunicamycin) (2.5 µg for 3 hrs), MYR (myriocin) (2.5 µg for 3 hrs) and 0.5M NaCl (10 min). B) Phosphorylation of CaHog1p requires the CaIre1p. Wild-type (PC6116), ire1Δ/ire1Δ (PC6144), and ire1Δ/pIRE1 (PC6145) cells were grown in YEPD and YEP-GAL medium (5.5 hrs). C) P∼Cek1p levels in the hog1Δ/hog1Δ mutant at 30°C and 37°C. Wild-type (PC6111) and hog1Δ/hog1Δ (PC5008) cells were grown in YEPD and YEP-GAL medium (5.5 hrs). D) Plate-washing assay of wild-type cells (PC6111) and the hog1Δ/hog1Δ mutant (PC5008) on YEP-GAL medium at 37°C for 48 hrs. The plate was photographed, washed, and photographed again to reveal invaded cells. E) P∼CaHog1p and P∼Cek1p levels in the cek1Δ/cek1Δ mutant at 30°C and 37°C. Wild-type (PC6111) and cek1Δ/cek1Δ (PC6114) cells were grown in YEPD and YEP-GAL medium (5.5 hrs). F) Model showing the roles of the HOG and filamentous growth pathways in the response to growth in galactose. Galactose is transported into cells and metabolized by genes under the control of Snf1p. As a result, metabolic respiration is increased, which by some mechanism (?) induces the UPR. Ire1p mediates activation of the HOG and filamentous growth pathways (Adhikari et al. SUBMITTED). The HOG and filamentous growth pathways induce different target genes to redundantly promote growth under this condition. The antagonistic roles of these pathways on each other's activities optimize the response.