Nitrogen-dependent calcineurin activation in the yeast Hansenula polymorpha
Highlights
► Calcineurin activation is regulated by nitrogen source quality in Hansenula polymorpha. ► Intracellular nitrate and Ca2+ trigger nitrate-assimilation gene expression. ► Central role of calcineurin and Ca2+ in nitrogen sensing.
Introduction
Calcium-mediated signalling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety of cellular processes, including gene expression. Fluctuations in the free cytosolic Ca2+ levels directly elicit a cellular response by altering the function of Ca2+ binding proteins and their targets (Uhlen and Fritz, 2010). Calcineurin is a Ca2+-calmodulin dependent serine/threonine protein phosphatase, highly conserved among mammals and yeast (Kuno et al., 1991). In Saccharomyces cerevisiae, calcineurin is a heterodimer containing a catalytic subunit, encoded by the functionally redundant genes CNA1 and CNA2, complexed with a regulatory subunit, the product of CNB1. Cells lacking the catalytic subunits, or the regulatory subunit, have no calcineurin activity. Calcineurin controls gene expression mainly through activation by dephosphorylation of the transcriptional factor Crz1 (Stathopoulos-Gerontides et al., 1999). Crz1 binds specifically to the calcineurin-dependent response element (CDRE) that is both necessary and sufficient to direct Ca2+-induced calcineurin-dependent gene expression (Stathopoulos and Cyert, 1997). In S. cerevisiae, calcineurin-deficient cells are sensitive to Na+, Li+ and Mn2+ (Farcasanu et al., 1995, Nakamura et al., 1993). Adaptation to high salt stress requires the presence of a plasma membrane Na+-ATPase involved in Na+ and Li+ efflux, Ena1. Cells deficient in calcineurin accumulate Na+ and Li+ due to decreased expression of ENA1 (Mendizabal et al., 2001). Moreover, calcineurin plays a role in regulating cell-wall structure (Zhao et al., 1998), Ca2+ pumps and exchangers responsible for Ca2+ homoeostasis (Cunningham and Fink, 1994, Cunningham and Fink, 1996), and recovery from pheromone-induced growth arrest (Moser et al., 1996). Unlike in S. cerevisiae, in the non-conventional yeast Torulaspora delbrueckii calcineurin is required for both Ca2+ and Mn2+ tolerance but the calcineurin-Crz1 pathway has no apparent role in Na+ homoeostasis (Hernandez-Lopez et al., 2006). In the case of Candida dubliniensis or Cryptococcus neoformans, calcineurin has been shown to be involved in drug tolerance, hyphal growth and virulence (Chen et al., 2011). In addition to the mentioned functions, the role of calcineurin in nutrient acquisition has been recently documented. Thus, calcineurin has also been linked with the utilization of inorganic phosphate in Aspergillus fumigatus (da Silva Ferreira et al., 2007). Moreover, in S. cerevisiae, the activation of Crz1 transcription factor was observed in de-repressed cells after adding glucose in the presence of extracellular calcium (Groppi et al., 2011).
NCR1 is the regulatory mechanism through which microorganisms respond to environmental nitrogen availability by utilizing preferred nitrogen sources such ammonium and glutamine instead of non-preferred ones (Cooper, 2002, Magasanik and Kaiser, 2002). In S. cerevisiae, NCR involves the localization of GATA transcription factors Gln3 and Gat1 outside the nucleus in the presence of preferred nitrogen sources (e.g. glutamine). This prevents the expression of genes related to the utilization of non-preferred nitrogen sources (e.g. proline). In this framework, the negative regulator Ure2 has been shown to be involved in cytoplasm localization of Gln3 (Beck and Hall, 1999, Cooper, 2002, Kulkarni et al., 2001). In spite of the capacity of S. cerevisiae to scavenge its environment for a variety of alternative non-preferred nitrogen sources, this yeast is not able to use nitrate, the most abundant inorganic nitrogen form in soils, as sole nitrogen source. For this reason, nitrate assimilation has mostly been studied in bacteria, filamentous fungi and plants, and more recently in the non-conventional yeast Hansenula polymorpha (renamed Pichia angusta and currently Ogataea angusta) thanks to the development of basic genetics tools (gene disruption, integrative vectors, transformation systems, etc.) (Brito et al., 1999, Faber et al., 1994, Gonzalez et al., 1999, Saraya et al., 2012) and the sequencing of its genome (Ramezani-Rad et al., 2003; http://genome.jgipsf.org/Hanpo2). Nitrate assimilation in this yeast follows the pathway described for plants and filamentous fungi. Nitrate enters the cell via a high-affinity nitrate transporter (Ynt1), then is reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) (Siverio, 2002). The genes encoding these proteins (YNT1, YNR1 and YNI1) are subjected to dual control: NCR, triggered by preferred nitrogen sources (e.g. ammonium, glutamine or glutamate) and specific induction mechanisms, elicited by nitrate itself. The mentioned genes are closely clustered with YNA1 and YNA2, encoding two highly similar Zn(II)2Cys6 transcriptional factors found to be indispensable for nitrate induction in H. polymorpha (Avila et al., 2002, Avila et al., 1998, Machin et al., 2004).
Recent evidence from our laboratory (Rodriguez et al., 2010) showed that calcineurin might be involved in nitrogen assimilation. We showed that Gat1 levels were regulated by calcineurin and that Ure2 was involved in extracellular Ca2+ entry, regulating the expression of PMR1 (encoding the P-type Ca2+-ATPase involved in Ca2+ transport ER2-Golgi) suggesting that nitrogen-assimilation gene expression could be, at least in part, dependent on calcineurin. These findings led us to outline a preliminary working model of calcineurin’s role in nitrogen assimilation (Rodriguez et al., 2010) (Fig. 1) and to hypothesize that exposure to nitrate or nitrogen starvation could trigger an increase in total cell calcium, which in turn would activate the calcineurin signalling pathway. Here we provide further evidence to support and expand our preliminary model, finding a clear relationship between nitrogen sources present in the medium, cell Ca2+ content and calcineurin activation, but also showing sound evidence of the involvement of calcineurin in both nitrate-assimilation gene de-repression and induction.
Section snippets
Yeast strains and growth conditions
The H. polymorpha strains used in this work are listed in Supplemental Table 1. All strains are derivatives of the NCYC495 leu2 ura3 strain. Yeast cells were grown with shaking at 37 °C in YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) or 0.17% (w/v) yeast nitrogen base (YNB Difco), without amino acids or ammonium sulphate, 2% (w/v) glucose (this is termed synthetic medium) plus the nitrogen source indicated in each case. To determine ENA1-lacZ or YNR1-lacZ expression by
Calcineurin regulates nitrate-assimilation gene expression
In H. polymorpha, the lack of calcineurin regulatory subunit gene HpCNB1 dramatically decreases the levels of the GATA factors involved in NCR: HpGat1 and HpGat2 (Rodriguez et al., 2010). This led us to investigate the role of calcineurin in non-preferred nitrogen sources assimilation in yeast using nitrate as reference. We tested the growth of Δcnb1 in the presence of chlorate, the chlorine analogue of nitrate, which is reduced by NR4 to the toxic chlorite (Cove, 1976, Kosola and Bloom, 1996).
Discussion
How eukaryotic organisms are able to sense preferred nitrogen sources versus non-preferred, leading to an appropriate expression of the genes involved in the assimilation of the nitrogen source(s) available, is a key question as yet not completely answered. Here we show that calcineurin is involved in nitrate assimilation in H. polymorpha, acting on both NCR and nitrate induction mechanisms.
Regarding NCR, we had already found that the GATA transcription factor Gat1 is regulated by calcineurin
Conclusions
We conclude that Ca2+ and calcineurin play a central role in nitrogen signalling and assimilation. Thus, the nitrogen source modulates total cell Ca2+ content and calcineurin activation, although calcineurin also regulates nitrogen assimilation genes.
Funding
This work was supported by Grants BFU2010-16192 from ‘‘Ministerio de Ciencia e Innovación’’ (MICINN, Spain) and PI 2008/338 from Gobierno de Canarias to JMS. CR was a recipient of predoctoral fellowships from ‘‘Agencia Canaria de Investigación e Innovación y Sociedad de la Información’’ (ACIISI).
Acknowledgments
Access to the H. polymorpha genome database provided by Rhein Biotech GmbH (Duesseldorf, Germany) is gratefully acknowledged. We are thankful to Guido Jones for proofreading the manuscript.
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Recipient of predoctoral fellowship from ACIISI, Gobierno de Canarias.