Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

<div><p>Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In <i>Saccharomyces cerevisiae</i>, 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 <i>Candida albicans</i> 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.</p></div

Gene expression profiling by RNA seq analysis and qPCR.

<p><b>A</b>) Genes induced by salt, tunicamycin (TUN), or galactose (GAL). All RNA seq comparisons are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004734#pgen.1004734.s007" target="_blank">Table S1</a>. <b>B</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 <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004734#pgen.1004734.s002" target="_blank">Fig. S2</a></b>. <b>C</b>) Heat map of genes induced by the indicated stresses. Common targets and targets unique to each stimulus is shown. Asterisk, target of ESR. <b>D</b>) qPCR of HOG pathway target mRNAs in wild type and the <i>pbs2</i>Δ mutant grown in glucose (GLU, YEPD) and galactose (GAL, YEP-GAL). Error bars indicate +/−S.E.M. of three independent experiments. Actin (<i>ACT1</i>) mRNA was used as a control. <b>E</b>) Activity of p8X<i>CRE-lacZ</i> in wild-type cells (PC313) and <i>pbs2</i>Δ mutant (PC5035) grown in YEPD (5.5 hr), YEP-GAL (5.5 hr), and YEPD+0.4 M KCl (30 min). <b>F</b>) qPCR of Ste12p target mRNAs in wild type (PC538) and the <i>ste12</i>Δ (PC2382) mutant grown in glucose (YEPD) and galactose (YEP-GAL). See panel D for details.</p

Comparison of HOG pathway activation by galactose and osmotic stress.

<p>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. <b>A</b>) 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>B</b>) Graph of P∼Hog1p levels under the indicated conditions, as determined by ImageJ analysis. <b>C</b>) 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. <b>D</b>) Extended time course of Hog1∼P during growth in galactose. <b>E</b>) 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. <b>F</b>) 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. <b>G</b>) 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 <i>ssk1</i>Δ (PC1523), <i>ssk2</i>Δ (PC6086), <i>ssk22</i>Δ (PC6085), <i>ssk2</i>Δ <i>ssk22</i>Δ (PC6031), <i>ste11</i>Δ (PC3861), <i>ste11</i>Δ <i>ssk1</i>Δ (PC2061), <i>pbs2</i>Δ (PC2053) and <i>hog1</i>Δ (PC6047) mutants were grown in YEP-GAL medium or YEPD medium containing 0.4M KCl for 5 min.</p

Strains used in this study.

<p>Strains used in this study.</p

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

<p><b>A</b>) Morphology of wild-type cells (PC538) and the <i>pbs2</i>Δ mutant (PC2053), grown on YEPD and YEP-GAL for 24 hrs. Bar, 5 microns. <b>B</b>) p<i>FRE-lacZ</i> reporter activity in wild-type cells (PC313) and the <i>pbs2</i>Δ mutant (PC5035) in YEP-GAL medium. <b>C</b>) Role of protein tyrosine phosphatases in P∼Hog1p activity in galactose. Wild-type cells (PC538), and the <i>ptp2</i>Δ (PC6156), <i>ptp3</i>Δ (PC6157) and <i>ptp2</i>Δ <i>ptp3</i>Δ double mutant (PC6158) were grown in YEPD and YEP-GAL media for 5.5 hrs. <b>D</b>) P∼Kss1p activity in wild-type cells and the <i>pbs2</i>Δ mutant (PC2053) grown in YEP-GAL medium over a time course as indicated. <b>E</b>) P∼Hog1p activity in the <i>kss1</i>Δ mutant (PC620) grown in YEP-GAL medium for the times indicated. <b>F</b>) qPCR showing the relative expression of <i>STE12</i> mRNA in the wild-type (PC538), <i>pbs2</i>Δ (PC2053) and <i>ste12</i>Δ (PC2382) mutant cells. Error bars indicate +/− standard error mean of three independent experiments. Actin (<i>ACT1</i>) mRNA was used as a control. <b>G</b>) Ste12p-HA protein levels in the wild-type and <i>pbs2</i>Δ strains. Hog1p levels by immunoblot analysis are also shown.</p

Primers used for qPCR in the study.

<p>Primers used for qPCR in the study.</p

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

<p><b>A</b>) Immunoblot showing P∼Hog1p levels in cells grown in glucose (YEPD), galactose (YEP-GAL) or glucose and galactose (YEPD+2% GAL). <b>B</b>) Wild-type cells (PC6016) and the <i>gal3</i>Δ, <i>gal4</i>Δ, <i>gal7</i>Δ and <i>gal10</i>Δ mutants grown in YEP-GAL. <b>C</b>) P∼Hog1 levels in cells grown under the indicated conditions for 3 h with or without antimycin, ANT. <b>D</b>) Wild type (PC538) and the <i>aco1</i>Δ (PC3912), <i>fum1</i>Δ (PC6152), <i>mdh1</i>Δ (PC6153) and <i>kgd1</i>Δ (PC6155) and <i>idh1</i>Δ (PC6154) mutants were grown in galactose for 5.5 hrs. <b>E</b>) Wild-type cells (PC538), and the <i>snf1</i>Δ (PC560), <i>mig1</i>Δ (PC4843) and <i>snf1</i>Δ <i>mig1</i>Δ (PC6076) mutants were grown in YEP-GAL medium for 5.5 hrs.</p

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.

<p><b>A</b>) Serial dilutions of wild-type (PC313), <i>ste7</i>Δ (PC4928), <i>pbs2</i>Δ (PC5035) and <i>ste7</i>Δ <i>pbs2</i>Δ (PC6272) cells were spotted on YEPD and YEP-GAL media. <b>B</b>) Morphology of wild-type cells (PC538), the <i>pbs2</i>Δ mutant (PC2053), the <i>ste7</i>Δ mutant (PC4982), and the <i>ste7</i>Δ <i>pbs2</i>Δ double mutant (PC6272) grown YEP-GAL media for 24 hrs. Bar, 5 microns. <b>C</b>) Septin staining of wild-type and <i>pbs2</i>Δ cells harboring the pCdc12p-GFP plasmid. Cells were grown to mid-log phase in YEPD. <b>D</b>) Mat formation in cells lacking the filamentous growth or HOG pathways. Wild-type (PC538), <i>flo11</i>Δ (PC1029), and <i>pbs2</i>Δ (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.</p

The role of Flo11 overexpression on upward growth in the plane of the Z-axis.

<p><b>A</b>) Microcolonies of wild-type cells (PC538) and cells overexpressing <i>FLO11</i> (PC2712) were examined by microscopy at 10× after 24 h incubation at 30°C. Wild type and <i>ste12</i> mats on high agar concentrations is also shown Bar = 100 microns. <b>B</b>) Contour mapping of z-stack rendering of the indicated microcolonies in panel 7A are shown. Bar = 30 microns. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s006" target="_blank">Supplemental Movies S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s007" target="_blank">S6</a>. <b>C</b>) Wild-type (PC538), <i>flo11</i>Δ (PC1029), and <i>GAL-FLO11</i> (PC2712) cells were spotted onto YEP-GAL medium (8%) agar atop nitrocellulose filters for 24 h at 30°C. Colonies were photographed in transmitted light. Bar = 1 cm. At right, separation of the <i>GAL-FLO11</i> mat from the surface using forceps. <b>D</b>) Microscopic examination of the mats in panel C. Bar = 200 microns.</p

<i>S. cerevisiae</i> forms filamentous mats.

<p><b>A</b>) Wild-type cells (PC313) were spotted 2 cm apart onto 0.3% agar media that contained (YEPD; top panel) or lacked (YEP; bottom panel) glucose. The YEPD plate was incubated for 4 days and photographed; the YEP plate for 15 days. Bar = 1 cm. <b>B</b>) Microscopic examination of perimeters of mats in 1A. Bar = 100 microns. <b>C</b>) The origin of filamentous mats. Wild type (PC538) cells were examined on synthetic medium either containing 2% glucose (SCD) or lacking glucose (SC) in 0.3% agar medium for 24 h at 30°C. A compiled Z-stack rendering of typical microcolonies are shown. Bar = 20 microns. <b>D</b>) Same strains in 1C were examined on rich medium either containing 2% glucose (YEPD) or lacking glucose (YEP) in 0.3% agar. A representative microscopic image is shown. Bar = 10 microns. E) Vegetative mats mature into filamentous mats over time as nutrients become limiting. Two mats of wild type (PC313) strain were spotted bilaterally (1.5 cm apart) on YEPD and YEP media (+0.2% galactose) containing 0.3% agar media. The number of filaments occurring along the circumference of mats was scored on a scale of 1, 2, or 3 dots at 20× magnification corresponding to 3, 6, or 9 filaments or greater, respectively. Dots were plotted on a circle representing the outline of one of the mats with right hemispheres corresponding to the side of the mat facing a second mat. Asymmetric filamentation observed in the right hemisphere of 2d, Glu can possibly result from nutritional stress compounded by nutrient depletion from adjacent mats. Filamentation was monitored and plotted after growth for 1, 2, 3, and 4 days. Quantitation of pseudohyphae was complicated at longer time points when biofilms began to variegate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Halme1" target="_blank">[60]</a>. Bar = 1 cm.</p