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

Knödler A, Konrad G, Mayinger P - BMC Mol. Biol. (2008)

Bottom Line: We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of SAC1 that is responsible for PI(4)P-mediated regulation.Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Nephrology and Hypertension, Oregon Health & Science University, Portland OR 97239, USA. andiknoedler@gmx.de

ABSTRACT

Background: 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.

Results: Here we show that the expression of lipid phosphatase Sac1p in the yeast Saccharomyces cerevisiae 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 SAC1 gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of SAC1 that is responsible for PI(4)P-mediated regulation. Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels.

Conclusion: 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.

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Elevated activity of the SAC1 promoter in a sac1 mutant background. (A) Diagram depicting a reporter construct used to examine expression activity in the yeast Saccharomyces cerevisiae. The 5'-UTR of SAC1 ranging from bp -500 to -1 was fused to the open reading frame of GFP. (B) Expression from the GFP reporter constructs. Wild-type and sac1Δ yeast cells transformed with a CEN-based plasmid containing the SAC1(-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. (C, D) Quantitation of relative GFP expression levels in wild-type, sac1Δ (C) and sac1-8 (D) strain backgrounds. Data are from at least three independent experiments (+/-SE).
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Figure 1: Elevated activity of the SAC1 promoter in a sac1 mutant background. (A) Diagram depicting a reporter construct used to examine expression activity in the yeast Saccharomyces cerevisiae. The 5'-UTR of SAC1 ranging from bp -500 to -1 was fused to the open reading frame of GFP. (B) Expression from the GFP reporter constructs. Wild-type and sac1Δ yeast cells transformed with a CEN-based plasmid containing the SAC1(-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. (C, D) Quantitation of relative GFP expression levels in wild-type, sac1Δ (C) and sac1-8 (D) strain backgrounds. Data are from at least three independent experiments (+/-SE).

Mentions: To identify the regulatory elements that are required for SAC1 gene transcription in the yeast Saccharomyces cerevisiae, we generated a reporter construct to examine SAC1 promoter activity. A genomic region comprising 500 bp upstream of the SAC1 open reading frame (SAC1-500/-1) was fused to the gene encoding green fluorescent protein (GFP) (Fig. 1A). The activity of the SAC1(-500/-1) 5'-UTR was then determined by monitoring intracellular GFP levels (Fig. 1B). Yeast cells in which the wild-type copy of SAC1 was deleted, showed a five-fold elevated expression from the reporter construct (Fig. 1B, C). The phosphatase-deficient sac1-8 mutant caused a similar degree of upregulated SAC1-GFP reporter activity (Fig 1D), suggesting that the SAC1 promoter is regulated by a mechanism that responds to a loss of Sac1p enzyme activity. To identify essential elements within the SAC1(-500/-1) 5'-UTR, we constructed a series of truncations within this region and assayed promoter activity (Fig 2A). Elimination of a 150-bp fragment containing a putative TATA box element (bp -50 to -46) within the 5'-UTR abolished expression (SAC1(-500/-150), Fig. 2A). Further truncations led to the discovery of a 100-bp element directly upstream of the SAC1 open reading frame that is necessary for promoter activity (Fig 2A). Significantly, the SAC1(-100/-1) minimal promoter was not only essential for gene transcription, but also sufficient for producing an elevated expression response in a sac1Δ background (Fig. 2B, C).


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

Knödler A, Konrad G, Mayinger P - BMC Mol. Biol. (2008)

Elevated activity of the SAC1 promoter in a sac1 mutant background. (A) Diagram depicting a reporter construct used to examine expression activity in the yeast Saccharomyces cerevisiae. The 5'-UTR of SAC1 ranging from bp -500 to -1 was fused to the open reading frame of GFP. (B) Expression from the GFP reporter constructs. Wild-type and sac1Δ yeast cells transformed with a CEN-based plasmid containing the SAC1(-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. (C, D) Quantitation of relative GFP expression levels in wild-type, sac1Δ (C) and sac1-8 (D) strain backgrounds. Data are from at least three independent experiments (+/-SE).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2258305&req=5

Figure 1: Elevated activity of the SAC1 promoter in a sac1 mutant background. (A) Diagram depicting a reporter construct used to examine expression activity in the yeast Saccharomyces cerevisiae. The 5'-UTR of SAC1 ranging from bp -500 to -1 was fused to the open reading frame of GFP. (B) Expression from the GFP reporter constructs. Wild-type and sac1Δ yeast cells transformed with a CEN-based plasmid containing the SAC1(-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. (C, D) Quantitation of relative GFP expression levels in wild-type, sac1Δ (C) and sac1-8 (D) strain backgrounds. Data are from at least three independent experiments (+/-SE).
Mentions: To identify the regulatory elements that are required for SAC1 gene transcription in the yeast Saccharomyces cerevisiae, we generated a reporter construct to examine SAC1 promoter activity. A genomic region comprising 500 bp upstream of the SAC1 open reading frame (SAC1-500/-1) was fused to the gene encoding green fluorescent protein (GFP) (Fig. 1A). The activity of the SAC1(-500/-1) 5'-UTR was then determined by monitoring intracellular GFP levels (Fig. 1B). Yeast cells in which the wild-type copy of SAC1 was deleted, showed a five-fold elevated expression from the reporter construct (Fig. 1B, C). The phosphatase-deficient sac1-8 mutant caused a similar degree of upregulated SAC1-GFP reporter activity (Fig 1D), suggesting that the SAC1 promoter is regulated by a mechanism that responds to a loss of Sac1p enzyme activity. To identify essential elements within the SAC1(-500/-1) 5'-UTR, we constructed a series of truncations within this region and assayed promoter activity (Fig 2A). Elimination of a 150-bp fragment containing a putative TATA box element (bp -50 to -46) within the 5'-UTR abolished expression (SAC1(-500/-150), Fig. 2A). Further truncations led to the discovery of a 100-bp element directly upstream of the SAC1 open reading frame that is necessary for promoter activity (Fig 2A). Significantly, the SAC1(-100/-1) minimal promoter was not only essential for gene transcription, but also sufficient for producing an elevated expression response in a sac1Δ background (Fig. 2B, C).

Bottom Line: We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of SAC1 that is responsible for PI(4)P-mediated regulation.Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Nephrology and Hypertension, Oregon Health & Science University, Portland OR 97239, USA. andiknoedler@gmx.de

ABSTRACT

Background: 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.

Results: Here we show that the expression of lipid phosphatase Sac1p in the yeast Saccharomyces cerevisiae 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 SAC1 gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of SAC1 that is responsible for PI(4)P-mediated regulation. Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus