Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate
© Knödler et al; licensee BioMed Central Ltd. 2008
Received: 04 October 2007
Accepted: 28 January 2008
Published: 28 January 2008
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.
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.
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.
Phosphorylated derivatives of phosphatidylinositol, collectively called phosphoinositides, play essential roles in a wide range of cellular processes situated at intracellular membranes . Recent evidence indicates that phosphoinositides are not only short-lived signals that activate downstream regulatory networks, but also play constitutive roles in organelle identity and membrane dynamics . A key property of individual phosphoinositides is their precisely regulated compartment-specific localization [2, 3]. The control and maintenance of diverse intracellular phosphoinositide pools is achieved through the functional interplay of specific sets of lipid kinases and phosphatases. Although it has been established that deficiencies in certain lipid phosphatases can lead to severe human disease , it is unknown as to how the expression of these enzymes is regulated. In contrast, the transcriptional regulation of enzymes involved in the biosynthesis of major membrane phospholipids is well characterized . The cellular concentrations of metabolic intermediates required for phospholipid biosynthesis, such as inositol, choline and phosphatidic acid, determine the levels of expression of their respective biosynthetic enzymes [6, 7]. However, whether the expression of lipid phosphatases and kinases is controlled by similar mechanisms remains unclear.
The polyphosphoinositide phosphatase Sac1p is a major regulator of PI(4)P levels at the endoplasmic reticulum (ER) and Golgi [8–10]. The precise distribution of PI(4)P between these two organelles is critical for coordinating cell growth with the secretory pathway . Here we show that the cellular levels of yeast Sac1p are regulated at the transcriptional level. We have identified a novel 9-bp element within the SAC1 promoter region that is necessary for the regulation of promoter activity. Furthermore, we demonstrate that intracellular levels of PI(4)P correlate with Sac1p protein levels.
Identification of promoter elements for regulation of SAC1 expression
SAC1 expression is regulated independent of inositol levels and ER stress
Sac1p plays an important role in ER-function by promoting ATP uptake and oligosaccharide biosynthesis [11, 15]. Disruption of SAC1 induces ER stress and causes constitutive activation of the unfolded protein response (UPR) . To test directly whether SAC1 expression is controlled by the UPR, we induced ER stress by treating cells with the reducing agent dithiothreitol (DTT) . While DTT triggered a substantial increase in the cellular levels of the ER chaperone Kar2p (Fig. 4D), expression from the SAC1(-500/-1) 5'-UTR did not change significantly (Fig. 4D). This result eliminates the possibility that SAC1 expression is under control of the UPR. sac1 mutants also display defects in actin cytoskeletal arrangement and are sensitive to drugs such as caffeine and Calcofluor White (CFW) [13, 14]. However, treating cells with CFW, an agent causing cell wall defects and thus activating the cell integrity pathway, had no obvious effect on SAC1 expression (data not shown).
Intracellular levels of PI(4)P correlate with SAC1 promoter activity
In yeast, many enzymes required for phospholipid biosynthesis show a common pattern of transcriptional regulation . Soluble and membrane-bound precursors for phospholipid biosynthesis such as inositol, choline and phosphatidic acid play a major role in this regulation [6, 7]. In contrast, little is known about the transcriptional regulation of enzymes controlling the cellular levels of the phosphorylated derivatives of these phospholipids. While Sac1p function is essential when yeast cells are deprived of inositol , the expression of SAC1 is not regulated by inositol itself. Instead Sac1p protein levels respond to the cellular levels of PI(4)P, which is the major substrate of this lipid phosphatase. PI(4)P is concentrated in distinct intracellular pools that have diverse yet essential cellular functions such as in regulating membrane trafficking and actin cytoskeletal organization [8, 10, 11]. In proliferating cells, Sac1p is responsible for turning over the PI(4)P that is generated by the PI 4-kinase Stt4p . We find that alterations in this Stt4p-specific PI(4)P pool are mechanistically linked to the control of SAC1 expression.
Membrane homeostasis and organellar traffic both rely on precisely regulated phosphoinositide gradients. In growing cells, Sac1p plays an important role in preventing random equilibration of PI(4)P at intracellular membranes, a phenotype commonly observed in sac1 mutants [8, 9]. Linking SAC1 expression to the levels of PI(4)P ensures that sufficient levels of the lipid phosphatase are continuously available to fulfill this task. Analysis of promoter elements required for this regulation revealed the partially palindromic 9-bp motif in the 5'-UTR of SAC1 that is critical for expression. Partial palindromic sequences have also been found in other cis-acting promoter elements . However, queries in the Saccharomyces cerevisiae promoter database (SCPD) indicate that the ACCACAGGT element does not overlap with any known consensus sequence for DNA binding proteins and therefore represents a novel motif. SAC1 promoters in higher eukaryotes have not yet been defined and it remains to be seen whether expression of the mammalian SAC1 homologs is regulated via a similar element.
sac1 mutants display accumulation of PI(4)P at the nuclear envelope and it is possible that nuclear phosphoinositides activate or recruit hitherto uncharacterized factors required for transcription. Recent reports indicated that phosphoinositides play important roles inside the nucleus and nuclear phosphoinositide-binding proteins have been discovered [27, 28]. While our results support the idea that PI(4)P is a direct regulator of SAC1 gene expression, it is also possible that a metabolite downstream of PI(4)P is the actual signal transducer. PI(4)P can be rapidly converted to PI(4,5)P2 by the PIP kinase Mss4p [29, 30]. However, sac1 mutant strains do not show elevated PI(4,5)P2 levels  and it is therefore unlikely that PI(4,5)P2 is directly involved in this regulation. Another potential mechanism could involve soluble inositol phosphate species. Both PI(4)P and PI(4,5)P2 can be hydrolyzed by phospholipase C giving rise to inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate respectively . These soluble signal transducers can be further phosphorylated in the nucleus where they are involved in transcriptional control and mRNA export [32, 33]. It remains to be determined whether these molecules play a role in regulating SAC1 expression and identifying the additional components of this signaling mechanism awaits further investigations.
This study characterizes a promoter element required for regulated expression of the lipid phosphatase Sac1p in yeast. This enzyme controls the distinct intracellular pools of PI(4)P required for membrane traffic and homeostasis. Distinct from phospholipid biosynthetic enzymes, whose expression is largely regulated by small soluble phospholipid precursors, the activity of the SAC1 promoter correlates with the intracellular levels of PI(4)P. We propose that the precise control of Sac1 protein levels by the membrane concentration of its major substrate ensures proper maintenance of organelle-specific phosphoinositide gradients.
Strains, reagents, and other procedures
Plasmids and yeast strains
CEN ARS URA3 GFP
CEN ARS URA3 SAC1 5' UTR (-500/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-242/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-170/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-500/-150)-GFP
CEN ARS URA3 SAC1 5' UTR (-125/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-83/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-114/-1)-GFP
CEN ARS URA3 SAC1 5' UTR (-100/-1)-GFP
CEN ARS URA3 SAC1 5' UTR Δ(-100/-84)-GFP
CEN ARS URA3 SAC1 5' UTR Δ(-83/-70)-GFP
CEN ARS URA3 SAC1 5' UTR Δ(-91/-84)-GFP
CEN ARS URA3 SAC1 5' UTR Δ(-100/-92)-GFP
MATαtrp1-delta901 leu2-3,112 his3-delta200 ura3-52 lys2-801 suc2-delta9 can1::hisG
MATa trp1-delta901 leu2-3,112 his3-delta200 ura3-52 lys2-801 suc2-delta9 can1::hisG sac1::TRP
MATa leu2-3, 112 ura3-52 his3-delta200 trp1-delta901 lys2-801 suc2-delta9 sac1::TRP1 stt4::HIS3 pSTT4-4 (LEU2 CEN6 stt4-4)
Pik1::ADE2-1 sac1::TRP YEplac181::pik1-12
MATαtrp1-delta901 leu2-3,112 his3-delta200 ura3-52 lys2-801 suc2-delta9 can1::hisG opi1::HIS3
MATa trp1-delta901 leu2-3,112 his3-delta200 ura3-52 lys2-801 suc2-delta9 can1::hisG sac1::TRP opi1::HIS3
Generation of SAC1 promoter constructs
Quantification of protein levels
Cells expressing GFP under the control of SAC1 5'-UTR constructs were grown in Hartwell's complete media (HC) supplemented with the appropriate amino acids and harvested in early logarithmic growth phase. 5 OD cells were collected, washed in water and resuspended in 200 μl 2× Laemmli buffer and 200 μl glass beads. Lysates were prepared by vortexing for one minute. Supernatants were boiled for 5 minutes and analyzed by SDS-PAGE and immunoblotting. Protein levels were measured by determination of band size and band density using NIH Image software (version 1.62). Protein amounts of GFP were normalized against protein amounts of glucose-6-phosphate dehydrogenase.
Since sac1 mutants are inositol auxotrophs, yeast cells were cultivated in 5.5 μM inositol prior to and during the labeling procedure. Early log phase cells were incubated with 10 μCi/ml myo- [3H]inositol for 2–3 doubling times. Labeling, extraction and deacylation of lipids was performed as described previously . HPLC analysis of glycerophosphoinositols was carried out on a 250 × 4.6-mm Partisil SAX column (Whatman, Florham Park, NJ) using a Jasco HPLC system equipped with an LB 508 Radioflow detector (Berthold, Bad Wildbach, Germany). Elution and quantification of glycerophosphoinositols were performed as described .
We thank Lieu Than for technical help. We also thank Suparna Kanjilal and Teresa Nicolson for comments on the manuscript. This work was funded by National Institute of Health grant GM071569 (P.M).
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