Direct Regulation of Genes Involved in Glucose Utilization by the Calcium/Calcineurin Pathway

Abstract

Failure to use glucose as carbon source results in transcriptional activation of numerous genes whose expression is otherwise repressed. HXT2 encodes a yeast high affinity glucose transporter that is only expressed under conditions of glucose limitation. We show that HXT2 is rapidly and potently induced by environmental alkalinization, and this requires both the Snf1 and the calcineurin pathways. Regulation by calcineurin is mediated by the transcription factor Crz1, which rapidly translocates to the nucleus upon high pH stress, and acts through a previously unnoticed Crz1-binding element (calcineurindependent response element) in the HXT2 promoter (؊507 GGGGCTG ؊501). We demonstrate that, in addition to HXT2, many other genes required for adaptation to glucose shortage, such as HXT7, MDH2, or ALD4, transcriptionally respond to calcium and high pH signaling through binding of Crz1 to their promoters. Therefore, calcineurin-dependent transcriptional regulation appears to be a common feature for many genes encoding carbohydrate-metabolizing enzymes. Remarkably, extracellular calcium allows growth of a snf1 mutant on low glucose in a calcineurin/Crz1-dependent manner, indicating that activation of calcineurin is sufficient to override a major deficiency in the glucose-repression pathway. We propose that alkalinization of the medium results in impaired glucose utilization and that activation of certain glucose-metabolizing genes by calcineurin contributes to yeast survival under this stress situation.Glucose is the major carbon and energy source for most cells, and it is by far the preferred carbon source for the budding yeast Saccharomyces cerevisiae. In this organism glucose uptake is the limiting step in the utilization of this sugar, and the relevance of glucose to yeast metabolism is highlighted by the unusually large number of hexose transporter genes present in its genome (see Refs. 1 and 2 for reviews). These proteins transport their substrates by passive, gradient-dependent, energyindependent diffusion. At least six of them (Hxt1-7) have been shown to act as glucose transporters, whereas others are considered to transport galactose (Gal2) or other hexoses (Hxt5 and Hxt8 -17) (1, 3).These diverse glucose transporters exhibit different kinetic characteristics, and each of them appears particularly suited for a specific circumstance. For instance, Hxt1 is a low affinity, high capacity transporter, whereas Hxt2, Hxt6, and Hxt7 are examples of high affinity glucose transporters. Experimental evidence indicates that a strain lacking Hxt1-7 (often denominated as hxt null mutant) is unable to grow on glucose or other hexoses such as fructose or mannose (4, 5). Expression of HXT2, HXT6, and HXT7 allows growth of the hxt strain on low (0.1%) glucose, whereas other transporters are unable to do so (6). This confirms the role of these genes as high affinity glucose transporters, even though the major physiological role for glucose uptake under glucose shortage conditions can be attributed to Hxt2 (2).The expression pat...

Calcineurin-Crz1 Signaling in Lower Eukaryotes

“…Finally, Crz1 is responsible for the transcription of several genes involved in glucose metabolism in S. cerevisiae, B. cinerea, and C. neoformans, including genes encoding hexose transporters and enzymes of glycolysis, the citric acid cycle, and alcohol, glycogen, or trehalose metabolism (23,33,60). For NFAT2, it has been shown that it activates the expression of glucagon (112).…”

“…1 (30), caffeine (31), temperature (32), pH (33), blue light (34), the nature of the nitrogen source (35), and antifungal drugs (36). All of these signals increase the intracellular Ca 2ϩ concentration.…”

“…Such Ca 2ϩ oscillations have already been known for a long time in D. discoideum (44) and in various cell lines (reviewed in reference 45). For mammalian cells, it has been shown that NFAT (together with other transcription factors) de- (23,33,60) Glucagon and insulin expression (112, 113) Ion homeostasis/osmoregulation (14,17,(19)(20)(21)(22)(23)(24)69) Osmoregulation (102)(103)(104) Nuclear localization Constant after blue-light treatment (34) Constant after ionomycin/Ca 2ϩ treatment (41) Kinases for rephosphorylation Glycogen synthase kinase GskA (19) Glycogen synthase kinase GSK3 (49) codes such calcium oscillations in a specific frequency range (45). It will therefore be of great interest in the future to see if Crz1 and its orthologues can also decode different frequencies of calcium oscillations.…”

Alkali Metal Cation Transport and Homeostasis in Yeasts

“…Northern analysis showed that PHO89 is transcribed under conditions of P i limitation, although it was shown later that it is also strongly induced by alkaline pH even when the cells are grown in medium with normal phosphate concentrations (256). Remarkably, induction of PHO89 at high pH seems to be largely dependent on calcineurin, and the gene also transcriptionally responds to increased extracellular calcium (246,280). Kinetic characterization of Pho89 showed that it has an apparent K m for phosphate of 0.5 M, has an optimum pH of 9.5, and is highly specific for Na ϩ ; activation of P i uptake is maximal at an Na ϩ concentration of 25 mM (156).…”

Assessing Differential Expression Measurements by Highly Parallel Pyrosequencing and DNA Microarrays: A Comparative Study

“…To this end, we have chosen a well-established perturbation model, the exposure of yeast cells to an abrupt increase in external pH. Previous DNA microarray-based analyses of the effect of this perturbation on gene expression remodeling have shown that it results in several hundred of genes being induced or repressed (Lamb and Mitchell, 2003;Lamb et al, 2001;Platara et al, 2006;Ruiz et al, 2008;Serrano et al, 2002Serrano et al, , 2006Viladevall et al, 2004), as a result of the modulation of diverse signaling pathways (for a recent review, see Arino, 2010). In this work we present a comparison of the transcriptional changes detected after short-term (10 min) exposure to high pH stress (pH = 8.0) for both wildtype and snf1 mutant strains.…”