Two phosphorylation sites in Ste12 are required for full transcriptional induction.

<p>A. Alignment of 2 tryptic phospho-peptides across 6 yeast species. Possible phosphorylation sites (serines and threonines) and minimal MAPK consensus sequences (S/T,P) are in bold. B. Western blots of lysate from yeast strains containing the indicated wild type or mutant Ste12, treated or not treated with 1 µM alpha factor for 15 minutes probed with anti-Ste12 and anti-GAPDH. 4x mut is Ste12^{S400A,S402A,T405A,S406A}. C. Histograms of the single cell (n > 2000 cells) pheromone response measured by p<i>PRM1</i>-mCherry fluorescence. Cells were treated with 4 nM alpha factor for 3 hrs followed by cycloheximide for 2 hrs, and measured by flow cytometry. D. Dose responses of cells treated with 12 concentrations of pheromone as described in C. Figure plots means of the unimodal distributions. Error bars depict the standard error of the mean. E. Cartoon model of the function of the phosphorylation of S400 and T525 F. Simulation of the ODE model across a dose response of pheromone.</p

Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

<div><p>Cell signaling systems transmit information by post-translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in <i>Saccharomyces cerevisiae</i>. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-protein coupled receptor (GPCR). We used published mass spectrometry-based proteomics data to identify putative sites of phosphorylation on pheromone pathway components, and we used evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of putative phosphorylation events that contribute to adjust the input-output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results suggest that relatively small quantitative influences from individual phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.</p> </div

Pheromone induced mating pathway in yeast.

<p>Pheromone (α factor) binds to a G-protein coupled receptor (Ste2). Binding promotes dissociation of the heterotrimeric G protein into Gα and Gβγ. Free Gβγ initiates downstream signaling by forming a complex with the scaffold protein Ste5 at the plasma membrane, as well as the small GTPase Cdc42, the adaptor protein Ste50 and the kinase Ste20. Ste5 further recruits the MAPK cascade members, Ste11, Ste7 and Fus3. Once this complex forms, Ste20 phosphorylates and activates Ste11; Ste11 then phosphorylates and activates Ste7; Ste7 phosphorylates and activates the MAPK Fus3. Dashed arrows indicate phosphorylation of proteins involved in the pheromone response shown here; solid arrows show connections to the high osmolarity response pathway, filamentation pathway, cell cycle control and polarized growth. Ste11 also participates in the hyper-osmotic response and the filamentation MAPK pathways, and Ste7 also participates in the filamentation pathway. Active Fus3 executes the different cellular responses to pheromone by phosphorylating and activating the transcriptional complex of Ste12, Dig1 and Dig2 to express the pheromone responsive target genes. In addition to transcriptional activation, Fus3 also arrests the cell cycle and initiates polarized growth. Figure omits other phosphorylation events and feedback. Phosphorylation events on the proteins Ste50, Dig1 and Ste12 (shaded in black) are the focus of this work.</p

Reporter Strains.

<p>a—Copy number was determined via qPCR, using <i>ama-1</i> as a control, detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s011" target="_blank">S1 Text</a>, Section 5.</p><p>Reporter Strains.</p

Phosphorylation of S202 on Ste50 inhibits pathway activity at low doses of pheromone.

<p>A. Alignment of a Ste50 tryptic phospho-peptide across 6 yeast species. Possible phosphorylation sites (serines and threonines) and minimal MAPK consensus sequences (S/T,P) are in bold. This peptide is not within either the SAM or the RA domains. B. Western blots of lysate from yeast strains containing the indicated wild type or mutant Ste50 that had been in the absence or presence of 1 µM alpha factor for 15 minutes probed with anti-Ste50 and anti-GAPDH. C. Histograms of the single cell pheromone response measured by p<i>PRM1</i>-YFP fluorescence. Cells (n > 300 cells) were treated with 1 nM alpha factor for 3 hrs followed by cycloheximide for 2 hrs, imaged by fluorescent microscopy, and quantified with Cell-ID. D. Dose responses of cells treated with 5 concentrations of pheromone as described in C. The means of the unimodal distributions are plotted. Error bars depict the standard error of the mean. E. Cartoon model of the function of the phosphorylation of S202. F. Simulation of the ODE model across a dose response of pheromone.</p

Layout of cells comprising the adult <i>C</i>. <i>elegans</i> intestine.

<p>Top panel shows the location of the left-handed helical half twist. Bottom panel shows the intestine within an animal. Anteriormost ring is comprised of four cells, remaining rings are made up of two cells, all arranged around a hollow core with the half twist between ring V and ring VI. Cells are identified by proper intestine cell name (e.g., int5L), progenitor cell (lineage, e.g., Ealp), and number (e.g., #12). Cells in L lineage are even numbers and cells in R lineage are odd numbers. We refer to these cells by their numbers in the x axis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.g004" target="_blank">Fig 4</a>. A more anatomically detailed cartoon with corresponding microscopic images that are also anatomically detailed is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s001" target="_blank">S1 Fig</a>; identification of the nuclei in the cells in the twist in microscopic images is also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s006" target="_blank">S6 Fig</a>.</p

Expression of two differently colored <i>P</i>_{<i>hsp-16</i>.<i>2</i>} reporters in the same animal using multicopy and single copy reporters.

<p>DIC micrographs and fluorescent images show the three posterior-most intestinal cell rings (rings VII-IX). In the figure, posterior is on the left and dorsal on the top. Top row shows an animal expressing two multicopy reporters. Bottom row shows an animal expressing two single copy reporters. In about 1/3 of the animals expressing two multicopy reporters, there is a dramatic difference in the expression of the reporters in at least one cell. The strong bias toward expression of the red allele in int9R (white arrow, top row, fourth panel) occurred in slightly more than half of the animals showing differential expression (12/21). We did not observe such pronounced expression bias in the intestine cells of the animals expressing the two single copy reporters (typical image shown in bottom row).</p

Interindividual variation in expression of <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporter strains by flow and by image cytometry.

<p>a—For flow measurements, numbers come from about 500 animals measured for each strain, repeated in ten runs on ten different days. CV is the Coefficient of Variation, or relative standard deviation (CV = Standard Deviation/Mean).</p><p>b—For microscopic measurements numbers come from the summed values of all intestine cells measured with image cytometry, which is a total of three measured CV values for each reporter.</p><p>c—Mean expression measured in flow. We could not compare mean expression data acquired by microscopy because we needed to increase the PMT detector gain for the single copy reporter.</p><p>d—Strain TJ3001.</p><p>e—Strain TJ375.</p><p>Interindividual variation in expression of <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporter strains by flow and by image cytometry.</p

Fluorescent proteins.

<p>A) Body plan of fluorescent proteins. DarkCitrine is fused to the N terminus of TFP and Citrine with a GSGG linker (black bar). B) Emission spectra from yeast expressing each XFP excited by a 458 nm laser and emission collected from 460–650 nm. (Double-headed arrows indicate wavelength bands used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109940#pone-0109940-g002" target="_blank">Figure 2</a>)</p

Relationship between copy number and expression in <i>P_hsp-16.2</i>-XFP reporter strains.

<p><b>A)</b> X axis is reporter diploid copy number; Y axis shows expression level in PMT counts. Solid line shows a Hill function with a Hill coefficient of 0.6 (a quantitative measure of the nonlinear increase in expression with increasing copy number), fit to the data with an R² of 0.97. For each strain, the expression data is the average of at least three flow experiments that quantified about 500 animals per experiment. <b>B)</b> Average expression level of a single copy <i>P_hsp-16.2</i><i>-GFP</i> reporter in homozygotes and heterozygotes. Error bars show S.E.M. We picked over 200 F1 hermaphrodites from each cross and measured 84 F1 heterozygotes and 49 F1 homozygotes in flow; additional details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s011" target="_blank">S1 Text</a>, Section 5: Strain Construction.</p