Pho91 is a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol in yeast cells. In this study, we have demonstrated the pleiotropic effects of the PHO91 gene knockout in the methylotrophic yeast Ogataea parapolymorpha (Hansenula polymorpha, Ogataea angusta). The content of both acid-soluble and acid-insoluble inorganic polyphosphate (polyP) in the ∆pho91 cells was slightly higher compared to the strain with wild-type PHO91, when the cells were cultivated on glucose. The pho91-Δ mutations both in O. parapolymorpha and in Saccharomyces cerevisiae diminished resistance to cadmium and increased resistance to manganese and peroxide stresses. The cells of the mutant strain of O. parapolymorpha were unable to consume methanol due to the lack of methanol oxidase activity. We speculate that these effects are associated with the inability of mutant cells to mobilize phosphate from the vacuolar pool and/or defects in the signaling pathways involving phosphate, polyP, and inositol polyphosphates.

Bach Institute of Biochemistry Research Center of Biotechnology RAS Moscow Russian Federation

Federal Research Center Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences Institute for Biological Instrumentation of the Russian Academy of Sciences Pushchino Russian Federation

Federal Research Center Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences Skryabin Institute of Biochemistry and Physiology of Microorganisms Pushchino Russian Federation

Folia Microbiol (Praha). 2023 Jun;68(3):491. doi: 10.1007/s12223-023-01043-1. PubMed

See more in PubMed

Agaphonov M, Alexandrov A (2014) Self-excising integrative yeast plasmid vectors containing an intronated recombinase gene. FEMS Yeast Res 14(7):1048–1054. https://doi.org/10.1111/1567-1364.12197 PubMed DOI

Agaphonov MO, Beburov MYu, Ter-Avanesyan MD, Smirnov VN (1995) A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha. Yeast 11(13):1241–1247. https://doi.org/10.1002/yea.320111304 PubMed DOI

Albi T, Serrano A (2016) Inorganic polyphosphate in the microbial world. Emerging roles for a multifaceted biopolymer. World J Microbiol Biotechnol 32(2):27. https://doi.org/10.1007/s11274-015-1983-2

Andreeva N, Ryazanova L, Zvonarev A, Trilisenko L, Kulakovskaya T, Eldarov M (2018) Inorganic polyphosphate in methylotrophic yeasts. Apply Microbiol Biotechnol 102:5235–5244 DOI

Andreeva N, Ryazanova L, Ledova L, Trilisenko L, Kulakovskaya T (2022) Stress resistance of Saccharomyces cerevisiae strains overexpressing yeast polyphosphatases. Stresses 2:17–25. https://doi.org/10.3390/Stresses2010002 DOI

Auesukaree C, Homma T, Tochio H, Shirakawa M, Kaneko Y, Harashima S (2004) Intracellular phosphate serves as a signal for the regulation of the PHO pathway in Saccharomyces cerevisiae. J Biol Chem 279(17):17289–17294. https://doi.org/10.1074/jbc.M312202200 PubMed DOI

Azevedo C, Saiardi A (2017) Eukaryotic phosphate homeostasis: the inositol pyrophosphate perspective. TIBS 42(3):219–231. https://doi.org/10.1016/j.tibs.2016.10.008 PubMed DOI

Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70(1):241–250 PubMed DOI

Canadell D, Gonzalez A, Casado C, Arino J (2015) Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae. Mol Microbiol 95(3):555–572. https://doi.org/10.1111/mmi PubMed DOI

Choi J, Rajagopa A, Xu YF, Rabinowitz JD, O'Shea EK (2017) A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae. PLoS One 12(5):e0176085. https://doi.org/10.1371/journal.pone.0176085

Culotta VC, Daly MJ (2013) Manganese complexes: diverse metabolic routes to oxidative stress resistance in prokaryotes and yeast. ARS 9(9):933–944. https://doi.org/10.1089/ars.2012.5093 DOI

Eskes E, Deprez MA, Wilms T, Winderickx J (2018) pH homeostasis in yeast; the phosphate perspective. Curr Genet 64(1):155–161. https://doi.org/10.1007/s00294-017-0743-2 PubMed DOI

Giuseppin MLF, van Eijk HMJ, Bes BCM (1988) Molecular regulation of methanol oxidase activity in continuous cultures of Hansenula polymorpha. Biotechnol Bioeng 32:577–583 PubMed DOI

Genu V, Gödecke S, Hollenberg CP, Pereira GG (2003) The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity. Eur J Biochem 270(11):2467–2475 PubMed DOI

Góth L (1991) A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 196(2–3):143–151. https://doi.org/10.1016/0009-8981(91)90067-m PubMed DOI

Gray MJ, Jakob U (2015) Oxidative stress protection by polyphosphate-new roles for an old enter player. Curr Opin Microb 24:1–6. https://doi.org/10.1016/j.mib.2014.12.004 DOI

Gruzman MB, Titorenko VI, Ashin VV, Lusta KA, Trotsenko YA (1996) Multiple molecular forms of alcohol oxidase from the methylotrophic yeast Pichia Methanolica. Biochemistry (Moscow) 61(12):1537–1544

Hürlimann HC, Stadler-Waibel M, Werner TP, Freimoser FM (2007) Pho91 is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae. Mol Biol Cell 18(11):4438–4445 PubMed DOI PMC

Jensen LT, Ajua-Alemanji M, Culotta VC (2003) The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis. J Biol Chem 278:42036–42040 PubMed DOI

Karginov AV, Fokina AV, Kang HA, Kalebina TS, Sabirzyanova TA, Ter-Avanesyan MD, Agaphonov MO (2018) Dissection of differential vanadate sensitivity in two Ogataea species links protein glycosylation and phosphate transport regulation. Sci Rep 8:16428. https://doi.org/10.1038/s41598-018-34888-5 PubMed DOI PMC

Kulakovskaya TV, Andreeva NA, Karpov AV, Sidorov IA, Kulaev IS (1999) Hydrolysis of tripolyphosphate by purified exopolyphosphatase of Saccharomyces cerevisiae cytosol: kinetic model. Biochemistry (Moscow) 64:990–993

Lonetti A, Szijgyarto Z, Bosch D, Loss O, Azevedo C, Saiardi A (2011) Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases. J Biol Chem 286:31966–31974. https://doi.org/10.1074/jbc.M111.266320 PubMed DOI PMC

Morrissette VA, Rolfes RJ (2020) The intersection between stress responses and inositol pyrophosphates in Saccharomyces cerevisiae. Curr Genet 66:901–910. https://doi.org/10.1007/s00294-020-01078-8

Mouillon JM, Persson BL (2006) New aspects on phosphate sensing and signaling in Saccharomyces cerevisiae. FEMS Yeast Res 6(2):171–176. https://doi.org/10.1111/j.1567-1364.2006.00036.x PubMed DOI

Ozimek P, Marten Veenhuis M, van der Klei IJ (2005) Alcohol oxidase: a complex peroxisomal, oligomeric flavoprotein. FEMS Yeast Res 5:975–983 PubMed DOI

Pinson B, Merle M, Franconi JM, Daignan-Fornier B (2004) Low affinity orthophosphate carriers regulate PHO gene expression independently of internal orthophosphate concentration in Saccharomyces cerevisiae. J Biol Chem 279(34):35273–35280. https://doi.org/10.1074/jbc.M405398200 PubMed DOI

Popova Y, Thayumanavan P, Lonati E, Agrochao M, Thevelein JM (2010) Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor. Proc Natnl Acad Sci USA 107:2890–2895 DOI

Potapenko E, Cordeiro CD, Huang G, Storey M, Wittwer C, Dutta AK, Jessen HJ, Starai VJ, Docampo R (2018) 5-Diphosphoinositol pentakisphosphate (5-IP PubMed DOI PMC

Rao NN, Gómez-García MR, Kornberg A (2009) Inorganic polyphosphate: essential for growth and survival. Ann Rev Biochem 78:605–647 PubMed DOI

Reddi AR, Jensen LT, Culotta VC (2009) Manganese homeostasis in Saccharomyces cerevisiae. Chem Rev 109:4722–4732 PubMed DOI PMC

Sabbagh Y (2013) Phosphate as a sensor and signaling molecule. Clin Nephr 79(1):57–65. https://doi.org/10.5414/CN107322 DOI

Sohn JH, Choi ES, Kim CH, Agaphonov MO, Ter-Avanesyan MD, Rhee JS, Rhee SK (1996) A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1. J Bacteriol 178(15):4420–4428. https://doi.org/10.1128/jb.178.15.4420-4428

Thomas MR, O’Shea EK (2005) An intracellular phosphate buffer filters transient fluctuations in extracellular phosphate levels. Proc Nat Acad Sci USA 102(27):9565–9570. https://doi.org/10.1073/pnas.0501122102 PubMed DOI PMC

Tomar P, Sinha H (2014) Conservation of PHO pathway in ascomycetes and the role of Pho84. J Biosci 39(3):525–536. https://doi.org/10.1007/s12038-014-9435-y PubMed DOI

Tomashevsky A, Kulakovskaya E, Trilisenko L, Kulakovskiy IV, Kulakovskaya T, Fedorov A, Eldarov M (2021) VTC4 Polyphosphate polymerase knockout increases stress resistance of Saccharomyces cerevisiae cells. Biology 10:487. https://doi.org/10.3390/biology10060487 PubMed DOI PMC

Trilisenko L, Zvonarev A, Valiakhmetov A, Penin AA, Eliseeva IA, Ostroumov V, Kulakovskiy IV, Kulakovskaya T (2019) The reduced level of inorganic polyphosphate mobilizes antioxidant and manganese-resistance systems in Saccharomyces cerevisiae. Cells 8(5):461. https://doi.org/10.3390/cells8050461 PubMed DOI PMC

Vagabov VM, Trilisenko LV, Kulakovskaya TV, Kulaev IS (2008) Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae. FEMS Yeast Res 8:877–882. https://doi.org/10.1111/j.1567-1364.2008.00420.x

Wild R, Gerasimaite R, Jung J-Y, Truffault V, Pavlovic I, Schmidt A, Saiardi A, Jessen HJ, Poirier Y, Hothorn M, Mayer A (2016) Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science 352(6288):986–990. https://doi.org/10.1126/science.aad9858 PubMed DOI

Wykoff DD, Rizvi AH, Raser JM, Margolin B, O’Shea EK (2007) Positive feedback regulates switching of phosphate transporters in S. cerevisiae. Mol Cell 27:1005–1013. https://doi.org/10.1016/j.molcel.2007.07.022 PubMed DOI PMC

Yurimoto H, Sakai Y (2009) Methanol-inducible gene expression and heterologous protein production in the methylotrophic yeast Candida boidinii. Biotechnol Appl Biochem 53:85–92. https://doi.org/10.1042/BA20090030 PubMed DOI

Yurimoto Y, Yasuyoshi Sakai Y, Kato N (2002) Chapter 5. Methanol metabolism. In: Gellissen G (eds) Hansenula polymorpha: Biology and Applications. Wiley‐VCH Verlag GmbH.  https://doi.org/10.1002/3527602356.ch5

Inability of Ogataea parapolymorpha pho91-Δ mutant to produce active methanol oxidase can be compensated by inactivation of the PHO87 gene