|
Article |
Address correspondence to Alonzo H. Ross, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Rm. 819, Worcester, MA 01605. Tel.: (508) 856-8016. Fax: (508) 856-8017. email: Alonzo.Ross{at}umassmed.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: PTEN; phosphatidylinositol kinase; phosphatidylinositol-3,4,5-trisphosphate; phosphatidylinositol-3-phosphatase; fission yeast
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although classes I, II, and III PI 3-kinases are widely expressed in metazoa, only a single PI 3-kinase gene, vps34, has been identified in yeast (Takegawa et al., 1995). Unlike the class I enzymes, vps34p synthesizes PI(3)P but not PI(3,4)P2 or PI(3,4,5)P3. PI(3)P is involved in the control of vesicle trafficking to the vacuole (Odorizzi et al., 2000). The failure to detect PI(3,4,5)P3 (or PI(3,4)P2) in yeast is consistent with the lack of a class I PI 3-kinase and has led to the assumption that no biosynthetic pathway for PI(3,4,5)P3 exists in fission or budding yeast. Our observation that the Schizosaccharomyces pombe ptn1p has high homology to the mammalian PI(3,4,5)P3 phosphatase, PTEN, led us to question this assumption. We find that ptn1p, like its mammalian orthologue, is a PI(3,4,5)P3 phosphatase. Ptn1 disrupted (ptn1) cells have levels of PI(3,4,5)P3 comparable to mammalian cells, display irregularly shaped vacuoles and are osmotically fragile. PI(3,4,5)P3 synthesis in S. pombe required vps34p and its3p, but not fab1p. These results suggest a novel biosynthetic pathway for PI(3,4,5)P3 that evolved before the appearance of class I PI 3-kinases.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Ptn1p affects phosphoinositide levels in vivo
To test the role of ptn1 in vivo, we prepared a yeast strain (ptn1) lacking ptn1 and then introduced a pREP1 ptn1p expression vector. These yeast strains were labeled with [3H]inositol, the lipids were extracted and deacylated, and the levels of phosphoinositide were analyzed by HPLC. The ptn1
cells had six- to eightfold increased levels of PI(3,4)P2 and PI(3,4,5)P3 as compared with wild-type cells (Fig. 2). Restoration of ptn1p levels with a ptn1p expression vector lowered PI(3,4)P2 and PI(3,4,5)P3 levels close to wild-type levels. Manipulation of ptn1p levels did not affect PI(3)P levels, indicating that in vivo PI(3)P is not a significant substrate for ptn1p, as has been suggested for human PTEN (Leslie and Downes, 2002). These results confirm that ptn1p is a PI(3,4,5)P3 phosphatase in vivo and provide the first report of PI(3,4,5)P3 in yeast.
|
|
Ptn1p affects vacuole morphology and osmotic fragility
The ptn1 cells grew normally and had a normal morphology by bright field microscopy. However, using EM, we found that the ptn1
cells had misshapen vacuoles (Fig. 4). To quantify this phenotype, we counted the cells with at least 50% irregularly shaped vacuoles. Fig. 4 C shows that >70% of the ptn1
cells presented this phenotype. These findings demonstrate an effect of ptn1 disruption on vacuole morphology. We analyzed the ptn1p subcellular localization with a pREP42 GFP-ptn1p expression vector. The GFP-ptn1p fusion protein was detected in both punctate structures (0.51.0 µm in diameter) and septa of dividing cells (Fig. 5 A). As controls, we expressed a pREP42-GFP vector or an untagged pREP1-ptn1p vector. We did not observe punctate or septal fluorescence.
|
|
Based on the lack of colocalization of GFP-ptn1p with rhodamine phalloidin, the punctate structures were not associated with actin patches (unpublished data). To further characterize these punctate structures, we performed immuno-EM, using anti-GFP antibodies. Clusters of immunogold particles were detected in association with vesicular structures (Fig. 5 C, arrow). The gold particles were generally not associated with the larger vacuoles. Based on the size, we suspect that these structures may be endosomes (Prescianotto-Baschong and Riezman, 2002). Control cells that did not express GFP-ptn1p did not show significant numbers of immunogold particles.
We next sought a PH domain protein that binds PI(3,4)P2 and/or PI(3,4,5)P3 and, thereby, mediates downstream signaling. The S. pombe genome includes 21 proteins with predicted PH domains (Wood et al., 2002). Based on rules developed for mammalian PI(3,4,5)P3-binding PH domains (Rameh et al., 1997; Lietzke et al., 2000), we identified seven candidates and tested them for phosphoinositide binding using filters spotted with lipids. Two of these PH domain proteins showed binding to PI(3,4,5)P3 in vitro, although none showed high specificity for binding to this lipid compared with PI(4,5)P2. The first was a predicted protein designated SPAC 11E3.11C, which is a homologue of the ARNO/cytohesin/Grp family of Arf exchange factors (Fig. 6 A). The second was ksg1p, which is the S. pombe homologue of the mammalian PI(3,4,5)P3 regulated kinase, PDK1 (Niederberger and Schweingruber, 1999; unpublished data). Although the in vivo binding specificity of lipid binding domains often correlates with this in vitro assay, this is not always the case (Yu et al., 2004). A more reliable assay is relocalization of the protein in vivo in response to a perturbation that alters phosphoinositide levels. In both wild-type and ptn1 cells, GFP-ksg1p showed septal and plasma membrane localization (unpublished data). A possible explanation is that the ksg1 PH domain targets the plasma membrane and septum via PI(4,5)P2 rather than PI(3,4,5)P3. In 83% (72/86) of ptn1
cells, the GFP-11E.11C protein localized to endosome-like structures, septa, and growing ends (Fig. 6 B), resembling the distribution of GFP-ptn1p (Fig. 5 A). In contrast, examination of >100 wild-type cells showed no clear localization of the GFP-11E3.11C protein (Fig. 6 C). These experiments establish 11E3.11C as a good candidate for a PI(3,4)P2/PI(3,4,5)P3binding PH domain protein in S. pombe.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The discovery of ptn1 led us to examine the biosynthetic pathway for PI(3,4,5)P3 synthesis. We discovered a novel pathway that originates with synthesis of PI(3)P by vps34p, followed by the conversion of PI(3)P to PI(3,4,5)P3. Its3p, the S. pombe orthologue of mammalian type I PIP 5-kinases, converts PI(3)P into PI(3,4)P2, as has been shown to occur for mammalian type I PIP 5-kinases (Zhang et al., 1997; Tolias et al., 1998). The enzyme that catalyzes the last step in the synthesis of PI(3,4,5)P3 has not been identified, but by analogy with the mammalian pathway, may also be its3p. The observation that wild-type cells have undetectable or very low levels of PI(3,4)P2 and PI(3,4,5)P3 indicates that, as for mammalian cells, these lipids are tightly regulated in fission yeast. This regulation may occur at the level of synthesis and/or degradation of these lipids. Here we show that the ptn1p has an important role in maintaining the low levels of PI(3,4)P2 and PI(3,4,5)P3 in S. pombe. Understanding the spatial and temporal regulation of PI(3,4)P2 and PI(3,4,5)P3 synthesis are important questions for future studies.
Ptn1p, like PI(3)P (Gillooly et al., 2000) and vps34p (Stack et al., 1995), was found associated with vesicular structures, and ptn1 cells show irregularly shaped vacuoles and are more readily lysed by osmotic stress. However, we also observed ptn1p associated with the septa of dividing cells. Hence, as in mammalian cells, PI(3,4,5)P3 (and/or PI(3,4)P2) in S. pombe likely has multiple functions, regulating different processes in different regions of the cell.
The mechanism by which this lipid affects cell function in fission yeast remains to be determined. One can imagine that, as for mammalian cells, PI(3,4,5)P3 (and/or PI(3,4)P2) may function to recruit target proteins to specific subcellular locations via binding to protein modules. The S. pombe genome includes 21 putative PH domains (Wood et al., 2002), which in mammalian cells bind to PI phosphates and mediate many of the downstream effects. Our investigation of the phosphoinositide binding specificity of S. pombe PH domains revealed that the GFP-11E3.11C PH domain has distinct subcellular distributions in wild-type and ptn1 cells, suggesting that it is regulated by PI(3,4)P2 and/or PI(3,4,5)P3. However, by the filter binding assay the 11E3.11C PH domain is not specific for PI(3,4)P2 or PI(3,4,5)P3. There are several possible explanations. First, there may be experimental complications, relating to incomplete folding of in vitro translated PH domains, thereby, compromising PH domain specificity. In addition, binding of PH domains to filters is an excellent method for surveying phosphoinositide specificity, but binding of PH domains to undiluted phosphoinositides on a filter is sometimes less selective than in biological membranes (Snyder et al., 2001). Second, specific binding of S. pombe PH domains to membranes might require interactions with both lipid and protein targets. Indeed, some S. cerevisiae PH domains require multiple interactions for membrane binding (Yu et al., 2004). Third, localization of the 11E3.11C PH domain in ptn1
cells may be due to a higher affinity for PI(3,4,5)P3 than PI(4,5)P2, as has been observed for the ARNO PH domain (Venkateswarlu et al., 1998; Cullen and Chardin, 2000). The 11E3.11C predicted protein is a homologue of the ARNO/cytohesin/Grp family and like these mammalian proteins, has an Arf GDP/GTP exchange domain and PH domain. Hence, PI(3,4,5)P3 in lower eukaryotes may act through a PH domain (domains) that binds multiple phosphoinositides, and PI(3,4,5)P3-specific PH domains may have evolved in more complex species.
In summary, the results presented here indicate that a pathway for the synthesis of PI(3,4,5)P3 from PI(3)P existed in yeast before the evolution of class I PI 3-kinases in higher eukaryotes, indicating a more ancient function for this important signaling molecule.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphatase assay
GST-PTEN and GST-ptn1 (1 µg/assay) were incubated with 25 nmoles of appropriate dioctanoyl PI substrates in 500 µl of assay buffer, containing 50 mM Tris-HCl, pH 7.5, and 2 mM DTT for 30 min at 37°C. The reaction was stopped by addition of malachite green solution (BIOMOL Research Laboratories, Inc.), and the enzyme activity was measured by the change in absorption at 650 nm using appropriate controls.
In vivo analysis of phosphoinositides
Log phase cultures of yeast strains were grown in Edinburgh minimal medium (EMM) synthetic media plus appropriate supplements. Cells were washed twice in inositol-free EMM medium and subcultured (106 cells/ml) for 20 h in 5 ml of the same medium containing 10 µCi of myo[2-3H]inositol. Labeled cells were harvested and lysed by vigorous vortexing with 0.5 ml 1 N HCl, 1 ml methanol-chloroform (1:1 vol/vol), and 1.5 g of acid-washed glass beads (Sigma-Aldrich). Bovine brain phosphoinositides (40 µg/sample; Sigma-Aldrich) were added as carrier lipid, and phase separation was induced by addition of 0.4 ml chloroform. The extracted lipids were deacylated and analyzed by anion exchange high pressure liquid chromatography using a Partisphere SAX column (Agilent Technologies), using an online detector (Serunian et al., 1991).
Microscopy
For localization of GFP-ptn1, cells were grown to early log phase in EMM+adenine+leucine+thiamine, washed twice in EMM+adenine+ leucine medium, and induced in the same medium for 20 h. For colocalization with actin, cells were fixed in 3.7% formaldehyde, stained with rhodamine phalloidin (Molecular Probes), and visualized with a Zeiss Axioskop and Apochromat 100X objective (n = 1.4). Micrographs were recorded with an AxioCam digital camera and OpenLab software (Improvision). For ultrastructural analysis, early log phase cells were fixed in 2% KMnO4 for 30 min at RT, washed and dehydrated in sequential grades of ethanol, embedded in Epon resin at 65°C overnight, and stained with lead citrate and uranyl acetate (Armstrong et al., 1993). Observation was based on examination of at least 100 cells. Digital images were prepared using Adobe Photoshop 7.0.
For immuno-EM, cells grown to log phase were fixed with 4% PFA plus 0.4% glutaraldehyde for 30 min at RT with shaking. The cells were then dehydrated and embedded in LR white resin. Sections were blocked in 2% BSA plus 0.2% normal goat serum. Sections were stained with anti-GFP mAb (1:100; Covance) or a polyclonal rabbit antibody (1:50; Abcam). After washing, the sections were incubated with 10 nm of anti-antibody gold particles (Jackson ImmunoResearch Laboratories). The samples were washed and fixed after with 2% glutaraldehyde. The sections were stained with 1% uranyl acetate plus 1% lead citrate. Finally, the sections were briefly exposed to osmium vapor to provide additional contrast.
Assay for osmotic integrity of yeast cells
Yeast cells were overlaid with 0.05 M glycine HCl, pH 9.5, 1% agar, and 10 mM 5-bromo-4-chloro-3-indolyl phosphate (Paravicini et al., 1992). The cells that lysed released alkaline phosphatase and turned bluish green.
Phosphoinositide binding by PH domains
Sequences containing the PH domains for ksg1 (residues 434-592), OBP1 (residues 254-350), OBP2 (residues 121-260), pob1 (residues 690-815), SPAC 11E311C (residues 500-942), SPAC 26A3.10 (residues 501-651), and SPBC 17G9.08C (residues 500-630) were in vitro transcribed and translated with [35S]methionine (Promega TNT coupled transcription/translation system). The 35S-labeled proteins were incubated in 3% (wt/vol) fatty acidfree BSA (Sigma-Aldrich), 0.05% (vol/vol) Tween 20, 150 mM NaCl, 50 mM Tris, pH 7.5, with PVDF membranes spotted with phospholipids. The synthetic diC16:0 phosphoinositides were from Cell Signals, Inc. and were spotted at 180, 60, and 20 pmoles per 100 nl spot. Phosphatidic acid was spotted at 180 and 60 pmoles. Ceramide, PI, sphingosine-1-phosphate, phosphatidylcholine, and phosphatidylethanolamine were each spotted at 180 pmoles. After incubation with 35S-labeled proteins for 2 h at 4°C, the membranes were washed with 0.05% Tween 20, 150 mM NaCl, 50 mM Tris, pH 7.5. Proteins binding to lipid spots were detected by phosphorimaging.
![]() |
Acknowledgments |
---|
This research was funded by National Institutes of Health grants to L.E. Rameh, D. McCollum, L.C. Cantley, and A.H. Ross.
Submitted: 26 April 2004
Accepted: 1 June 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armstrong, J., M.W. Craighead, R. Watson, S. Ponnambalam, and S. Bowden. 1993. Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator. Mol. Biol. Cell. 4:583592.[Abstract]
Cantley, L.C. 2002. The phosphoinositide 3-kinase pathway. Science. 296:16551657.
Cullen, P.J., and P. Chardin. 2000. Membrane targeting: what a difference a G makes. Curr. Biol. 10:R876R878.[CrossRef][Medline]
Gary, J.D., A.E. Wurmser, C.J. Bonangelino, L.S. Weisman, and S.D. Emr. 1998. Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis. J. Cell Biol. 143:6579.
Gillooly, D.J., I.C. Morrow, M. Lindsay, R. Gould, N.J. Bryant, J.M. Gaullier, R.G. Parton, and H. Stenmark. 2000. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19:45774588.
Halstead, J.R., M. Roefs, C.D. Ellson, S. D'Andrea, C. Chen, C.S. D'Santos, and N. Divecha. 2001. A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress. Curr. Biol. 11:386395.[CrossRef][Medline]
Heymont, J., L. Berenfeld, J. Collins, A. Kaganovich, B. Maynes, A. Moulin, I. Ratskovskaya, P.P. Poon, G.C. Johnston, M. Kamenetsky, et al. 2000. TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathway and plays a role in the developmental process of sporulation. Proc. Natl. Acad. Sci. USA. 97:1267212677.
Hinchliffe, K., and R. Irvine. 1997. Inositol lipid pathways turn turtle. Nature. 390:123124.[CrossRef][Medline]
Lee, J.-O., H. Yang, M.-M. Georgescu, A. Di Cristofano, T. Maehama, Y. Shi, J.E. Dixon, P. Pandolfi, and N.P. Pavletich. 1999. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell. 99:323334.[Medline]
Leslie, N.R., and C.P. Downes. 2002. PTEN: the down side of PI 3-kinase signalling. Cell. Signal. 14:285295.[CrossRef][Medline]
Li, J., C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis, L. Rodgers, R. McCombie, et al. 1997. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 275:19431947.
Lietzke, S.E., S. Bose, T. Cronin, J. Klarlund, A. Chawla, M.P. Czech, and D.G. Lambright. 2000. Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains. Mol. Cell. 6:385394.[Medline]
Maehama, T., and J.E. Dixon. 1998. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-triphosphate. J. Biol. Chem. 273:1337513378.
Maehama, T., G.S. Taylor, and J.E. Dixon. 2001. PTEN and myotubularin: novel phosphoinositide phosphatases. Annu. Rev. Biochem. 70:247279.[CrossRef][Medline]
Morishita, M., F. Morimoto, K. Kitamura, T. Koga, Y. Fukui, H. Maekawa, I. Yamashita, and C. Shimoda. 2002. Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe. Genes Cells. 7:199215.
Niederberger, C., and M.E. Schweingruber. 1999. A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation. Mol. Gen. Genet. 261:177183.[CrossRef][Medline]
Odorizzi, G., M. Babst, and S.D. Emr. 2000. Phosphoinositide signaling and the regulation of membrane trafficking in yeast. Trends Biochem. Sci. 25:229235.[CrossRef][Medline]
Paravicini, G., M. Cooper, L. Friedli, D.J. Smith, J.L. Carpentier, L.S. Klig, and M.A. Payton. 1992. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol. Cell. Biol. 12:48964905.[Abstract]
Prescianotto-Baschong, C., and H. Riezman. 2002. Ordering of compartments in the yeast endocytic pathway. Traffic. 3:3749.[CrossRef][Medline]
Rameh, L.E., A. Arvidsson, K.L. Carraway III, A.D. Couvillon, G. Rathbun, A. Crompton, B. VanRenterghem, M.P. Czech, K.S. Ravichandran, S.J. Burakoff, et al. 1997. A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains. J. Biol. Chem. 272:2205922066.
Serunian, L.A., K.R. Auger, and L.C. Cantley. 1991. Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol. 198:7887.[Medline]
Simpson, L., and R. Parsons. 2001. PTEN: life as a tumor suppressor. Exp. Cell Res. 264:2941.[CrossRef][Medline]
Snyder, J.T., K.L. Rossman, M.A. Baumeister, W.M. Pruitt, D.P. Siderovski, C.J. Der, M.A. Lemmon, and J. Sondek. 2001. Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins. J. Biol. Chem. 276:4586845875.
Stack, J.H., D.B. DeWald, K. Takegawa, and S.D. Emr. 1995. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J. Cell Biol. 129:321334.[Abstract]
Steck, P.A., M.A. Pershouse, S.A. Jasser, W.K.A. Yung, H. Lin, A.H. Ligon, L.A. Langford, M.L. Baumgard, T. Hattier, T. Davis, et al. 1997. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15:356362.[Medline]
Takegawa, K., D.B. DeWald, and S.D. Emr. 1995. Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology. J. Cell Sci. 108:37453756.
Tolias, K.F., L.E. Rameh, H. Ishihara, Y. Shibasaki, J. Chen, G.D. Prestwich, L.C. Cantley, and C.L. Carpenter. 1998. Type I phosphatidylinositol-4-phosphate 5-kinases synthesize the novel lipids phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 5-phosphate. J. Biol. Chem. 273:1804018046.
Venkateswarlu, K., P.B. Oatey, J.M. Tavare, and P.J. Cullen. 1998. Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr. Biol. 8:463466.[Medline]
Wood, V., R. Gwilliam, M.A. Rajandream, M. Lyne, R. Lyne, A. Stewart, J. Sgouros, N. Peat, J. Hayles, S. Baker, et al. 2002. The genome sequence of Schizosaccharomyces pombe. Nature. 415:871880.[CrossRef][Medline]
Yu, J.W., J.M. Mendrola, A. Audhya, S. Singh, D. Keleti, D.B. DeWald, D. Murray, S.D. Emr, and M.A. Lemmon. 2004. Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell. 13:677688.[CrossRef][Medline]
Zhang, X., J.C. Loijens, I.V. Boronenkov, G.J. Parker, F.A. Norris, J. Chen, O. Thum, G.D. Prestwich, P.W. Majerus, and R.A. Anderson. 1997. Phosphatidylinositol-4-phosphate 5-kinase isozymes catalyze the synthesis of 3-phosphate-containing phosphatidylinositol signaling molecules. J. Biol. Chem. 272:1775617761.
Zhang, Y., R. Sugiura, Y. Lu, M. Asami, T. Maeda, T. Itoh, T. Takenawa, H. Shuntoh, and T. Kuno. 2000. Phosphatidylinositol 4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast. J. Biol. Chem. 275:3560035606.