Characterization of Sro1, a novel stress responsive protein in Schizosaccharomyces pombe

The large amount of available genome sequencing data presents a huge challenge in the form of orphan sequences. This study reports the detailed functional characterization of one such orphan sequence in Schizosaccharomyces pombe. We identified this gene as a prominently upregulated 1.4 kb transcript in a screen for Cigarette smoke extract responsive genes in S. pombe and named it Stress Responsive Orphan 1 (Sro1). We report various functions of Sro1 in regulation of cellular behaviour under stress conditions. We show that this gene (Sro1) responds to a variety of stress conditions and that the expression of the gene is regulated mainly through the stress activated protein kinase (SAPK) Sty1 and its downstream transcription factor Atf1. Deletion of Sro1 also significantly alters the reactive oxygen species (ROS) generation profiles and the cell-cycle progression of S. pombe during stress conditions. The stress-specific alteration of the ROS generation profiles and checkpoint activation resulting from deletion of the gene suggest that Sro1 might be a key player in determining cellular responses/fate under stress conditions

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake

Regulation of Mat Responses by a Differentiation MAPK Pathway in Saccharomyces cerevisiae

Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK) pathway and the same cell adhesion molecule (Flo11) but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis) and substrate invasion (downward in the plane of the Z-axis), which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis) in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake

<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

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

The role of the MAPK pathway in regulating mat architecture when exposed to surfaces of different rigidities.

<p><b>A</b>) Contour maps in the Z-axis of wild type (PC538) mats incubated in media of different agar concentrations for 14d. Insets show mat morphology (left, photograph, bar, 1 cm; right, photomicrograph, bar, 200 microns) in 4% agar. The numbers in parentheses represent the average mat dry weight from two experiments with standard deviation shown. Scale bars for the X and Y-axes are in mm. <b>B</b>) Mats formed by a <i>ste12</i>Δ mutant (PC539) on different agar concentrations. Analysis is as described for panel A.</p