The Snf1 Protein Kinase Controls the Induction of Genes of the Iron Uptake Pathway at the Diauxic Shift in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression. In this study, we characterized a novel family of five genes whose expression is induced during the diauxic shift. These genes, FET3, FTR1, TIS11, SIT1, and FIT2, are involved in the iron uptake pathway. We showed that their induction at the diauxic shift is positively controlled by the Snf1/Snf4 kinase pathway. The transcriptional factor Aft1p, which is known to control their induction in response to iron limitation, is also required for their induction during the diauxic shift. The increase of the extracellular iron concentration does not affect this induction, indicating that glucose exhaustion by itself would be the signal. The possibility that the Snf1/Snf4 pathway was also involved in the induction of the same set of genes in response to iron starvation was considered. We demonstrate here that this is not the case. Thus, the two signals, glucose exhaustion and iron starvation, use two independent pathways to activate the same set of genes through the Aft1p transcriptional factor. In Saccharomyces cerevisiae the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression. In this study, we characterized a novel family of five genes whose expression is induced during the diauxic shift. These genes, FET3, FTR1, TIS11, SIT1, and FIT2, are involved in the iron uptake pathway. We showed that their induction at the diauxic shift is positively controlled by the Snf1/Snf4 kinase pathway. The transcriptional factor Aft1p, which is known to control their induction in response to iron limitation, is also required for their induction during the diauxic shift. The increase of the extracellular iron concentration does not affect this induction, indicating that glucose exhaustion by itself would be the signal. The possibility that the Snf1/Snf4 pathway was also involved in the induction of the same set of genes in response to iron starvation was considered. We demonstrate here that this is not the case. Thus, the two signals, glucose exhaustion and iron starvation, use two independent pathways to activate the same set of genes through the Aft1p transcriptional factor. Eukaryotic cells develop many processes to adapt to environmental changes. The yeast Saccharomyces cerevisiae is able to grow upon several carbon sources, among which glucose remains the favorite one. The use of glucose by fermentation leads to the production of ethanol in the medium. Then ethanol can be used after an adaptive process known as the diauxic shift, which is a transition from fermentative growth to respiratory growth. At this stage cells stop to divide, and the enzymatic equipment is modified to allow assimilation of carbon and production of energy from ethanol. This process involves a global change of gene expression. These changes have been monitored at the levels of proteome (1Boucherie H. J. Bacteriol. 1985; 161: 385-392Crossref PubMed Google Scholar, 2Bataille N. Thoraval D. Boucherie H. Electrophoresis. 1988; 9: 774-780Crossref PubMed Scopus (21) Google Scholar, 3Bataille N. Regnacq M. Boucherie H. Yeast. 1991; 7: 367-378Crossref PubMed Scopus (26) Google Scholar) and transcriptome (4DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3707) Google Scholar). As glucose is depleted, transcription of genes involved in gluconeogenesis, the tricarboxylic acid cycle, the glyoxylate cycle, and respiration is enhanced, whereas expression of genes involved in glycolysis and protein synthesis is decreased. The extent of these changes raises the question of which regulatory mechanisms lead to a coordinated expression of the genome in response to glucose depletion. The problem can be addressed by investigating the involvement of a particular transcription factor. Another possible method can be the alteration of transduction systems that transfer the signal of glucose starvation to the transcription factors. In both cases identification of up- and down-regulated genes can be achieved by using global approaches. In this way the role of the transcriptional activator Cat8p during the diauxic shift has been defined by the identification of its target genes (5Haurie V. Perrot M. Mini T. Jeno P. Sagliocco F. Boucherie H. J. Biol. Chem. 2001; 276: 76-85Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). It appears that although this factor is essential for growth on ethanol as the carbon source, only a limited part of the genes induced during the diauxic transition belongs to the Cat8p regulon. Cat8p is involved in the Snf1p-dependent pathway that is required in the response to glucose starvation. The protein kinase Snf1p is the catalytic subunit of a complex containing a regulator subunit Snf4p and a third element that can be Gal83p, Sip1p, or Sip2p. This heterotrimeric complex is the homologue of the mammalian AMP-activated protein kinase (for review, see Ref. 6Hardie D.G. Biochem. Soc. Symp. 1999; 64: 13-27PubMed Google Scholar). Like its mammalian homologue, Snf1p is activated in response to glucose limitation, a process that requires its own phosphorylation by an upstream kinase and its interaction with the regulatory subunit Snf4p (7McCartney R.R. Schmidt M.C. J. Biol. Chem. 2001; 276: 36460-36466Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The activated form of Snf1p phosphorylates target proteins including transcriptional activators, such as Cat8p and Sip4p, and the repressor Mig1p (8Carlson M. Curr. Opin. Microbiol. 1999; 2: 202-207Crossref PubMed Scopus (461) Google Scholar). The aim of the present study was to characterize new functions of the Snf1p-dependent pathway in the transcriptional changes occurring at the diauxic shift. We compared the transcriptional profiles of wild type and snf4Δ strains at the time of glucose exhaustion by using a genome-wide scan. We showed that genes involved in iron uptake are subject to a positive control by the Snf1 pathway in response to glucose starvation. This induction requires the transcriptional activator Aft1p. The results presented here suggest a link between the regulation of iron uptake and utilization and the adaptation to the respiratory growth during the diauxic transition. Strains and Culture Conditions—The wild type strains S. cerevisiae FY5 (MATα, mal) and FY2 (MATα, mal, ura3–52) derived from S288C were kindly provided by F. Winston (Harvard Medical School). The SNF4, CAT8, SNF1, and AFT1 deletions were constructed in the wild type strains FY5 or FY2 by replacing the gene with the kanMX4 cassette (9Wach A. Yeast. 1996; 12: 259-265Crossref PubMed Scopus (705) Google Scholar). The different yeast strains obtained are named YP3 (MATα, mal, cat8Δ::KanMX4), YP4 (MATα, mal, snf4Δ::KanMX4), YP11 (MATα, mal, snf1Δ::KanMX4), YP14 (MATα, mal, ura3–52, snf4Δ::KanMX4), YP17 (MATα, mal, aft1Δ::KanMX4), and YP31 (MATα, mal, ura3–52, snf1Δ::KanMX4). The single chromosomal integration of KanMX4 at these loci was controlled by PCR or Southern blot analyses. The strains expressing Aft1-HA 1The abbreviation used is: HA, hemagglutinin. were created by the transformation of pRS416-AFT1-HA, generously supplied by Y. Yamaguchi-Iwai (10Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), to FY2, YP14, and YP31. Cultures were performed at 22 °C in a 500-ml Erlenmeyer flask containing 50 ml of supplemented minimal medium YNBS (0.17% yeast nitrogen base without ammonium sulfate and amino acids, 0.5% ammonium sulfate, 2% glucose, 25 μg/ml inositol, 85 mm succinate/NaOH, pH 5.8) or complete medium YM1 (0.5% yeast extract, 0.17% yeast nitrogen base without ammonium sulfate and amino acids, 2% glucose, 1% Bacto-peptone, 0.5% ammonium sulfate, 85 mm succinate/NaOH, pH 5.8). To test the response to iron limitation, cultures were performed in the same conditions as mentioned above but in rich media YPD (1% yeast extract, 2% Bacto-peptone, 2% glucose) supplemented or not with the iron chelator ferrozine to a final concentration of 1 mm. Cultures were shaken at 360 rpm, and growth was monitored by measuring their absorbance at 600 nm (an A 600 of 1 corresponds to 2.4 × 107 cells/ml). Glucose Measurement—Glucose measurements were performed with a Sigma Diagnostic kit (catalog number 510-A). RNA Isolation—Total RNA was extracted as previously described (11Lindquist S. Nature. 1981; 293: 311-314Crossref PubMed Scopus (267) Google Scholar). Mini-array Filter Hybridization—Mini-array analyses were performed using genomic filters (Research Genetics) as described (5Haurie V. Perrot M. Mini T. Jeno P. Sagliocco F. Boucherie H. J. Biol. Chem. 2001; 276: 76-85Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Two independent experiments of comparison were performed with a single set of membranes. Between hybridizations, the efficiency of stripping (≥95%) was checked by scanning the filters. To allow comparison between separate filter hybridizations, the intensity of each spot was normalized to the total hybridization signal intensity. A gene was considered as potentially Snf4p-dependent when the spot displayed at least a 2-fold difference between the mutant and the wild type strains in the two experiments. Northern Blot Hybridization—15 μg of each RNA preparation was electrophoresed under denaturing conditions (12Lehrach H. Diamond D. Wozney J.M. Boedtker H. Biochemistry. 1977; 16: 4743-4751Crossref PubMed Scopus (2402) Google Scholar) and blotted onto nylon membranes (13Sambrook J. Fritschand E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Hybridization probes used were PCR-amplified with appropriate primers (sequences available upon request). Hybridization signals were detected using a Molecular Dynamics PhosphorImager, and quantification was achieved with the ImageQuant software (Molecular Dynamics). Preparation of Protein Extracts and Immunoblot Analysis—Proteins extraction was performed as previously described (14Pinson B. Kongsrud T.L. Ording E. Johansen L. Daignan-Fornier B. Gabrielsen O.S. Nucleic Acids Res. 2000; 28: 4665-4673Crossref PubMed Scopus (21) Google Scholar). Samples of 2.4 × 108 cells were harvested by centrifugation, rinsed with water, and suspended in 500 μl of 5% trichloroacetic acid before the addition of 500 mg of glass beads. Cells were disrupted by vortexing for 15 min at room temperature, and supernatants were transferred to clean tubes, incubated for 15 min on ice, and centrifuged for 5 min at 12,000 × g. Supernatants were discarded. Pellets were rinsed with ethanol 95%, neutralized, and dissolved in 50 μl of 1 m Tris-HCl, pH 8.0, and 125 μl of Laemmli buffer (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and then incubated for 3 min at 100 °C. Samples were cooled at room temperature and centrifuged before electrophoresis. Protein extracts were separated by SDS-PAGE using 6% polyacrylamide gel, transferred to polyvinylidene difluoride membrane (ICN), and analyzed by immunoblotting using anti-HA monoclonal antibodies (clone 12CA5, Berkeley Antibody Co., Richmond, CA). Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Eurogentec) followed by enhanced chemiluminescence with ECL Plus™ reagents 2.4 × 108 cells were dissolved in 50 μl of m pH 1 mm Cells were disrupted with glass and then μl of were used for each in a final μl of buffer were with or without of and Samples were incubated for min at and the was by the addition of 25 μl of Laemmli buffer and then incubated for 3 min at 100 °C before electrophoresis. of in snf4Δ complex is required for growth on carbon To genes under the control of the Snf1/Snf4 pathway during the diauxic shift, we to gene expression of a a under the conditions used in medium containing 2% glucose as carbon source, was that the growth and the of glucose of the snf4Δ were to of the wild type during the to glucose exhaustion In under the same the mutant displayed a alteration in growth The growth and the glucose of before glucose indicating that the of this is before the diauxic shift. In a rich medium this was but not were obtained from cells harvested 25 min after glucose depletion. Extracts obtained from wild and mutant cells were used to to filters. or genes were at least 2-fold in the mutant As Cat8p target genes were among these the genes in the mutant we five genes to the iron FTR1, FET3, FIT2, SIT1, and These five genes are known to be induced under conditions of iron in an Y. R. A. J. 1996; PubMed Scopus Google Scholar, T. J. J. Brown P.O. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. Brown P.O. D. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, F. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of expression at the diauxic shift in of Snf4p that be of the Snf1p-dependent pathway and that Aft1p this in the at the transcriptome was performed from extracts of cells harvested at a time of 25 min after the glucose Thus, we the possibility that between wild type and mutant were present before the diauxic transition. To this question we compared the profiles of levels in wild type and snf4Δ strains from min before glucose exhaustion to min after from this we that Snf4p is to the expression of the five genes to the of the transcriptome study, and we showed that these genes are induced through the diauxic shift. This induction is with glucose genes known as Aft1p target genes were at least 2-fold in the This be to the of We performed Northern experiments to expression of and Aft1p at the diauxic transition the expression of to be we were to of the and the genes by Northern blot at this whereas is possible to under iron we not test the expression of each Aft1p target gene by Northern that not of the are induced at the diauxic shift. This to Glucose snf4Δ is to use ethanol after glucose exhaustion we iron in the medium not The of expression of genes to the in this mutant be a of its to adapt to ethanol To this expression of was monitored in a The gene a transcriptional activator essential for the growth on ethanol. In this expression of is induced at the diauxic shift at the same in the wild type not Thus, is not the growth on ethanol that the induction of the in snf4Δ The is induced under conditions of iron The induction of Aft1p target genes at the diauxic shift be to an iron starvation with the glucose starvation. Thus, the expression of and was in an medium but supplemented with of At this the of growth of a on ethanol is not when iron in the the induction of and was detected when glucose the of induction was the same with or without iron addition In the induction of Aft1p target genes at the diauxic shift is independent to extracellular iron and appears to be a response to the glucose occurring at this stage of the The a Snf1p role of Snf4p is as a positive regulator of the kinase of To the Snf4p-dependent induction of the is also the of and in wild type and strains at the diauxic shift were compared A rich medium was used as we mentioned the growth of wild type and strains are in this medium. In the wild type the induction of and was and the use of rich medium not the of this In in the the levels of these two genes were not enhanced at the time of glucose We from this that the protein kinase Snf1p is essential in the Snf4p-dependent which leads to the induction of the Aft1p target genes at the diauxic transition. Thus, the in a is by a in kinase of The five genes FTR1, FET3, FIT2, SIT1, and to the regulon. Aft1p transcription under iron as its to a and its are in an (10Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Y. R. A. J. 1996; PubMed Scopus Google Scholar). To test the involvement of Aft1p during the diauxic shift, we the expression of and in a when glucose is was not detected in the mutant and the of induction of was compared with the in the wild type the transcriptional activator Aft1p is required for the induction of these two genes at the diauxic shift, and is involved in the pathway. of Aft1p at the and showed that Aft1p was during the diauxic transition. We to this in and A wild type FY2 was with a expressing an Aft1p Cells were from harvested at different before and during the diauxic shift. The of Aft1p on SDS-PAGE was by using an anti-HA Cells harvested before the glucose exhaustion form of Aft1p glucose this form and cells at least form of Aft1p. extracts from cells harvested after glucose exhaustion were with in of this form of Aft1p was not detected This that the of Aft1p is a phosphorylation that Snf1p is for the phosphorylation of several transcriptional such as Cat8p or we can that Aft1p be a target of this The same performed with extracts from snf4Δ cells an the same of Aft1p was in mutant and in wild type strains A and Thus, Snf4p is not for the phosphorylation of the protein Aft1p, with glucose depletion. This that Aft1p is not a of the Snf1p To this we the phosphorylation of Aft1-HA in a of the growth of this in a minimal we performed this from cells in a rich medium a difference in the of of the form of Aft1-HA to the glucose concentration between wild type and which be to a of glucose in the mutant appears that Aft1-HA is when glucose in of the kinase We can from these results the phosphorylation of Aft1p at the the diauxic in the same time as the induction of its target Snf4p Snf1p is required for the phosphorylation of Aft1p. Thus, Aft1p is not a of the kinase displayed by the complex The of the link between Snf1p kinase and by Aft1p remains to be is in the and snf4Δ the phosphorylation of Aft1p is not to the regulon. The Snf1 in the to Aft1p transcriptional factor the iron in response to iron We showed that genes to this iron were induced during the diauxic shift in a This response at the time of glucose exhaustion not change to the iron We the of this the involvement of the Snf1 pathway in the regulation of gene expression in response to iron starvation when glucose is available in the medium. To this yeast strains were in medium containing the growth iron was by the addition of the iron chelator ferrozine in the medium at a final concentration of 1 mm. The induction of by the iron starvation was monitored by Northern blot in wild type and In the wild type as as in the two mutant the is in cells harvested during growth. It is enhanced after the addition of ferrozine in the medium in the the of induction is for the wild type and for the two The induction in the wild type after the addition of ferrozine is by the with sulfate at a final concentration of mm. This that the of expression is not by the ferrozine itself but by the iron In a complex is not required for the induction of in response to iron is an essential for It is a in a of processes such as In the the of iron to the of a for the B. in and 2nd ed. Press, Scholar). Thus, iron to be factor of this regulation is the transcriptional regulator Aft1p. conditions of iron Aft1p the expression of several genes including involved in the uptake and Y. R. A. J. 1996; PubMed Scopus Google Scholar, Y. A. J. PubMed Scopus Google Scholar). We showed here that several Aft1p target genes, FET3, FTR1, TIS11, SIT1, and FIT2, are induced during the diauxic shift. This induction when glucose in the media is at the the diauxic transition. This induction is positively controlled by a and Aft1p is It appears that activated at the diauxic shift, would positively Aft1p These results the question of the of the link between the Snf1p kinase and the Aft1p transcriptional factor. In with the M. R. E. Yeast. 1997; PubMed Scopus Google Scholar), we showed that Aft1p is during the of the diauxic shift. We showed that this phosphorylation at the time of glucose exhaustion is with the induction of This that this be a for Aft1p possibility would be that the Snf1p kinase be of this we showed that Snf1p Snf4p is for the phosphorylation of Aft1p. the induction of Aft1p requires a this is not to genes was that Aft1p is in the and in conditions of iron This is for the transcription of FTR1, a gene to the Aft1p (10Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). It is not known in response to glucose exhaustion is controlled in the same that is the Snf1p be involved at this Another would be that Snf1p the expression of genes by an for by an of these the Snf1p-dependent pathway is involved in the regulation of transcriptional at different the of Mig1p is controlled in a Snf1p-dependent M. Biol. 1997; PubMed Scopus Google Scholar, J. H. J. Biochem. PubMed Scopus Google Scholar, S. M. Biol. PubMed Scopus Google Scholar), and Snf1p a role in the control of the transcriptional activator Cat8p at the of transcription and at the of A. A. E. B. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar). This link between complex and Aft1p can be with a S. A. 2000; PubMed Scopus (143) Google Scholar) that Aft1p target genes are under a control by of the protein kinase A of the yeast proteins kinase Glucose is associated with a in the to the of protein kinase A. This of is required for the from growth on carbon to growth on carbon diauxic E. D. Perrot M. Boucherie H. M. 1996; PubMed Scopus Google Scholar). these results also suggest a link between and the pathway. Another question is is the induction of iron uptake genes at the the diauxic The induction of these genes that cells have to to an of In that the signal does not to be a of extracellular We showed that the concentration of iron in the media such that a does not stop after glucose exhaustion has on the possible is that the signal from the It has been that the major of iron in the is the and also be the for iron F. E. R.R. 1988; PubMed Scopus Google Scholar). cells are from glucose to ethanol the iron is for and the iron and the iron and be for of iron J.L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These two proteins are associated in a The of this complex the of the to from fermentative to respiratory has been that the use of the membrane a and induction 5 min of and under indicating that a of the expression of these J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these we can that when cells the diauxic shift be an for the and the of the iron or the iron be a signal for the induction of genes for iron uptake the iron for induction of the be at the diauxic shift. results that not of the are induced at the diauxic shift we were to of the and the genes at this stage by Northern to this was The Snf1/Snf4 pathway would not control the Aft1p but an which would control a different set of we showed that the Snf1 pathway is not involved in the response to iron in the induction of the regulon. Thus, two different signals, iron and glucose starvation, can a which is the induction of the iron uptake by the transcriptional activator Aft1p through We F. Winston for yeast strains and Y. Yamaguchi-Iwai for