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).
- Di Paolo G, De Camilli P: Phosphoinositides in cell regulation and membrane dynamics. Nature 2006, 443: 651-7. 10.1038/nature05185.View ArticlePubMedGoogle Scholar
- Behnia R, Munro S: Organelle identity and the signposts for membrane traffic. Nature 2005, 438: 597-604. 10.1038/nature04397.View ArticlePubMedGoogle Scholar
- De Matteis MA, Di Campli A, Godi A: The role of the phosphoinositides at the Golgi complex. Biochim Biophys Acta 2005, 1744: 396-405. 10.1016/j.bbamcr.2005.04.013.View ArticlePubMedGoogle Scholar
- Pendaries C, Tronchere H, Plantavid M, Payrastre B: Phosphoinositide signaling disorders in human diseases. FEBS Lett 2003, 546: 25-31. 10.1016/S0014-5793(03)00437-X.View ArticlePubMedGoogle Scholar
- Carman GM, Henry SA: Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes. Prog Lipid Res 1999, 38: 361-99. 10.1016/S0163-7827(99)00010-7.View ArticlePubMedGoogle Scholar
- Jesch SA, Zhao X, Wells MT, Henry SA: Genome-wide analysis reveals inositol, not choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in yeast. J Biol Chem 2005, 280: 9106-18. 10.1074/jbc.M411770200.PubMed CentralView ArticlePubMedGoogle Scholar
- Loewen CJ, Gaspar ML, Jesch SA, Delon C, Ktistakis NT, Henry SA, Levine TP: Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 2004, 304: 1644-7. 10.1126/science.1096083.View ArticlePubMedGoogle Scholar
- Tahirovic S, Schorr M, Mayinger P: Regulation of intracellular phosphatidylinositol-4-phosphate by the Sac1 lipid phosphatase. Traffic 2005, 6: 116-30. 10.1111/j.1600-0854.2004.00255.x.View ArticlePubMedGoogle Scholar
- Roy A, Levine TP: Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p. J Biol Chem 2004, 279: 44683-9. 10.1074/jbc.M401583200.View ArticlePubMedGoogle Scholar
- Foti M, Audhya A, Emr SD: Sac1 lipid phosphatase and stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology. Mol Biol Cell 2001, 12: 2396-411.PubMed CentralView ArticlePubMedGoogle Scholar
- Faulhammer F, Konrad G, Brankatschk B, Tahirovic S, Knodler A, Mayinger P: Cell growth-dependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p. J Cell Biol 2005, 168: 185-91. 10.1083/jcb.200407118.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu J, Zhang MQ: SCPD: a promoter database of the yeast Saccharomyces cerevisiae. Bioinformatics 1999, 15: 607-11. 10.1093/bioinformatics/15.7.607.View ArticlePubMedGoogle Scholar
- Hughes WE, Pocklington MJ, Orr E, Paddon CJ: Mutations in the Saccharomyces cerevisiae gene SAC1 cause multiple drug sensitivity. Yeast 1999, 15: 1111-24. 10.1002/(SICI)1097-0061(199908)15:11<1111::AID-YEA440>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Cleves AE, Novick PJ, Bankaitis VA: Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function. J Cell Biol 1989, 109: 2939-2950. 10.1083/jcb.109.6.2939.View ArticlePubMedGoogle Scholar
- Kochendorfer KU, Then AR, Kearns BG, Bankaitis VA, Mayinger P: Sac1p plays a crucial role in microsomal ATP transport, which is distinct from its function in Golgi phospholipid metabolism. Embo J 1999, 18: 1506-15. 10.1093/emboj/18.6.1506.PubMed CentralView ArticlePubMedGoogle Scholar
- Tahirovic S, Schorr M, Then A, Berger J, Schwarz H, Mayinger P: Role for lipid signaling and the cell integrity MAP kinase cascade in yeast septum biogenesis. Curr Genet 2003, 43: 71-8.PubMedGoogle Scholar
- Whitters EA, Cleves AE, McGee TP, Skinner HB, Bankaitis VA: SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast. J Cell Biol 1993, 122: 79-94. 10.1083/jcb.122.1.79.View ArticlePubMedGoogle Scholar
- Bachhawat N, Ouyang Q, Henry SA: Functional characterization of an inositol-sensitive upstream activation sequence in yeast. A cis-regulatory element responsible for inositol-choline mediated regulation of phospholipid biosynthesis. J Biol Chem 1995, 270: 25087-95. 10.1074/jbc.270.42.25087.View ArticlePubMedGoogle Scholar
- Greenberg ML, Reiner B, Henry SA: Regulatory mutations of inositol biosynthesis in yeast: isolation of inositol-excreting mutants. Genetics 1982, 100: 19-33.PubMed CentralPubMedGoogle Scholar
- Ashburner BP, Lopes JM: Regulation of yeast phospholipid biosynthetic gene expression in response to inositol involves two superimposed mechanisms. Proc Natl Acad Sci USA 1995, 92: 9722-6. 10.1073/pnas.92.21.9722.PubMed CentralView ArticlePubMedGoogle Scholar
- Kohno O, Normington K, Sambrook J, Gething MJ, Mori K: The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol Cell Biol 1993, 13: 877-890.PubMed CentralView ArticlePubMedGoogle Scholar
- Guo S, Stolz LE, Lemrow SM, York JD: SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases. J Biol Chem 1999, 274: 12990-5. 10.1074/jbc.274.19.12990.View ArticlePubMedGoogle Scholar
- Audhya A, Foti M, Emr SD: Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics. Mol Biol Cell 2000, 11: 2673-89.PubMed CentralView ArticlePubMedGoogle Scholar
- Konrad G, Schlecker T, Faulhammer F, Mayinger P: Retention of the yeast Sac1p phosphatase in the endoplasmic reticulum causes distinct changes in cellular phosphoinositide levels and stimulates microsomal ATP transport. J Biol Chem 2002, 277: 10547-54. 10.1074/jbc.M200090200.View ArticlePubMedGoogle Scholar
- Faulhammer F, S Kanjilal-Kolar, Knodler A, Lo J, Lee Y, Konrad G, Mayinger P: Growth Control of Golgi Phosphoinositides by Reciprocal Localization of Sac1 Lipid Phosphatase and Pik1 4-Kinase. Traffic 2007.Google Scholar
- Mori K, Ogawa N, Kawahara T, Yanagi H, Yura T: Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in Saccharomyces cerevisiae. J Biol Chem 1998, 273: 9912-20. 10.1074/jbc.273.16.9912.View ArticlePubMedGoogle Scholar
- Bunce MW, Bergendahl K, Anderson RA: Nuclear PI(4,5)P(2): a new place for an old signal. Biochim Biophys Acta 2006, 1761: 560-9.View ArticlePubMedGoogle Scholar
- Deleris P, Gayral S, Breton-Douillon M: Nuclear Ptdlns(3,4,5)P3 signaling: an ongoing story. J Cell Biochem 2006, 98: 469-85. 10.1002/jcb.20695.View ArticlePubMedGoogle Scholar
- Homma K, Terui S, Minemura M, Qadota H, Anraku Y, Kanaho Y, Ohya Y: Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis. J Biol Chem 1998, 273: 15779-86. 10.1074/jbc.273.25.15779.View ArticlePubMedGoogle Scholar
- Desrivieres S, Cooke FT, Parker PJ, Hall MN: MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae. J Biol Chem 1998, 273: 15787-93. 10.1074/jbc.273.25.15787.View ArticlePubMedGoogle Scholar
- Flick JS, Thorner J: Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol 1993, 13: 5861-76.PubMed CentralView ArticlePubMedGoogle Scholar
- York JD, Odom AR, Murphy R, Ives EB, Wente SR: A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999, 285: 96-100. 10.1126/science.285.5424.96.View ArticlePubMedGoogle Scholar
- Odom AR, Stahlberg A, Wente SR, York JD: A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 2000, 287: 2026-9. 10.1126/science.287.5460.2026.View ArticlePubMedGoogle Scholar
- Sikorski RS, Hieter P: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989, 122: 19-27.PubMed CentralPubMedGoogle Scholar
- Harlow E, Lane D: Antibodies: A laboratory manual. Cold Spring Harbor Laboratory; 1988.Google Scholar
- Schorr M, Then A, Tahirovic S, Hug N, Mayinger P: The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase. Curr Biol 2001, 11: 1421-6. 10.1016/S0960-9822(01)00449-3.View ArticlePubMedGoogle Scholar