Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate

<p>Abstract</p> <p>Background</p> <p>Phosphoinositides play a central role in regulating processes at intracellular membranes. In yeast, a large number of phospholipid biosynthetic enzymes use a common mechanism for transcriptional regulation. Yet, how the expression of genes encoding lipid kinases and phosphatases is regulated remains unknown.</p> <p>Results</p> <p>Here we show that the expression of lipid phosphatase Sac1p in the yeast <it>Saccharomyces cerevisiae </it>is regulated in response to changes in phosphatidylinositol-4-phosphate (PI(4)P) concentrations. Unlike genes encoding enzymes involved in phospholipid biosynthesis, expression of the <it>SAC1 </it>gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of <it>SAC1 </it>that is responsible for PI(4)P-mediated regulation. Upregulation of <it>SAC1 </it>promoter activity correlates with elevated levels of Sac1 protein levels.</p> <p>Conclusion</p> <p>Regulation of Sac1p expression via the concentration of its major substrate PI(4)P ensures proper maintenance of compartment-specific pools of PI(4)P.</p

Protection of Armadillo/β-Catenin by Armless, a Novel Positive Regulator of Wingless Signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94's positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate-4

<p><b>Copyright information:</b></p><p>Taken from "Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate"</p><p>http://www.biomedcentral.com/1471-2199/9/16</p><p>BMC Molecular Biology 2008;9():16-16.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2258305.</p><p></p>beled with [H]myo-inositol. Phosphoinositides were extracted, deacylated and quantified by HPLC. Data are from three independent experiments (+/-SE).promoter activity in Δ and Δ PI 4-kinase double mutants. Yeast cells were transformed with a -based plasmid containing the (-500/-1)-GFP fusion construct and grown to early log phase at 33°C. Cell extractswere analyzed by SDS-PAGE and immunoblotting. Relative GFP expression levels were quantified. Data are from at least three independent experiments (+/-SE). Correlation of increased Sac1 protein levels and PI(4)P phosphatase deficiency. Wild-type and Δ yeast expressing either a myc-tagged wild-type Sac1p or phosphatase-deficient mutant myc-Sac1-22p from the promoter were grown to early log phase at 30°C. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate-1

<p><b>Copyright information:</b></p><p>Taken from "Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate"</p><p>http://www.biomedcentral.com/1471-2199/9/16</p><p>BMC Molecular Biology 2008;9():16-16.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2258305.</p><p></p> GFP in a -based vector. The plasmids were introduced into a wild-type strain background and promoter activity determined by measurement of relative GFP expression levels in cell extracts. Expression of the GFP reporter. Wild-type and Δ yeast cells transformed with a -based plasmid containing the (-100/-1)-GFP fusion construct were grown to early log phase at 30°C. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies. Quantitation of relative GFP expression levels. Data are from at least three independent experiments (+/-SE)

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate-2

<p><b>Copyright information:</b></p><p>Taken from "Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate"</p><p>http://www.biomedcentral.com/1471-2199/9/16</p><p>BMC Molecular Biology 2008;9():16-16.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2258305.</p><p></p> GFP in a -based vector. Expression of the GFP reporter. The respective plasmids were introduced into a wild-type strain background and promoter activity was determined by measuring relative GFP expression levels. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate-7

<p><b>Copyright information:</b></p><p>Taken from "Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate"</p><p>http://www.biomedcentral.com/1471-2199/9/16</p><p>BMC Molecular Biology 2008;9():16-16.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2258305.</p><p></p> GFP in a -based vector. Expression of the GFP reporter. The respective plasmids were introduced into a wild-type strain background and promoter activity was determined by measuring relative GFP expression levels. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate-0

<p><b>Copyright information:</b></p><p>Taken from "Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate"</p><p>http://www.biomedcentral.com/1471-2199/9/16</p><p>BMC Molecular Biology 2008;9():16-16.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2258305.</p><p></p> 5'-UTR of ranging from bp -500 to -1 was fused to the open reading frame of GFP. Expression from the GFP reporter constructs. Wild-type and Δ yeast cells transformed with a -based plasmid containing the (-500/-1)-GFP fusion construct were grown to early log phase at 30°C. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies. Quantitation of relative GFP expression levels in Δ and strain backgrounds. Data are from at least three independent experiments (+/-SE)

Protection of Armadillo/β-Catenin by Armless, a Novel Positive Regulator of Wingless Signaling

<div><p>The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94's positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated.</p></div