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* This work was supported by National Science Foundation Grant MCB-9807786 (to Z. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Iron is an essential nutrient. Its deficiency hinders the synthesis of ATP and DNA. We report that galactose metabolism is defective when iron availability is restricted. Our data support this connection because 1) galactose-mediated induction of GAL promoter-dependent gene expression was diminished by iron limitation, and 2) iron-deficient mutants grew slowly on galactose-containing medium. These two defects were immediately corrected by iron replacement. Inherited defects in human galactose metabolism are characteristic of the disease called galactosemia. Our findings suggest that iron-deficient galactosemic individuals might be more severely compromised than iron-replete individuals. This work shows that iron homeostasis and galactose metabolism are linked with one another.
Iron acts as an enzyme cofactor and protein structural component in living organisms. It is indispensable for an array of metabolic functions, including cellular energy production and DNA synthesis (
). Because iron is essential and toxic, its intracellular content must be tightly controlled.
Normal iron homeostasis requires the presence of copper, which is also an essential nutrient. High-affinity iron uptake in the yeast Saccharomyces cerevisiae is Cu2+-dependent because Fet3p, a component of the high-affinity iron transporter complex, is a Cu2+-dependent ferrous oxidase (
Because copper influences iron homeostasis, we hypothesized that iron might in turn affect copper homeostasis. To find if iron influences copper uptake, we compared the level of Ctr1p under iron deficiency and iron excess. Because CTR1 transcription was unaffected by changes in iron conditions (
). Our investigation serendipitously led us to discover that GAL gene expression is inhibited when iron is limited and that galactose is not metabolized in naturally iron-deficient cells. This defect is reminiscent of the inability of a galactosemic to metabolize galactose (
). The deletions were verified by PCR. Strains BY4741, BY4741aft1Δ, BY4741fet3Δ, and BY4741gal7Δ were kindly provided by Dennis Winge and Grant Hartzog.
Effects of Iron Deficiency on Galactose Metabolism
Iron Chelation and Galactose Induction of GAL1 Promoter—Cells bearing the plasmid p414GAL-CTR1myc were grown overnight in synthetic complete medium lacking tryptophan (SC-TRP) with raffinose (2%) in the place of glucose. Cells were reinoculated into medium of the same composition with or without bathphenathroline disulfonate (BPS)
) at a starting A600 of 0.4. Galactose (0.5%) was added, and cells were grown until an A600 of 0.8–1.0 was attained. Yeast whole cell extracts were prepared, and Ctr1-Myc was detected by Western blotting using anti-Myc antibodies (
AFT1/FET3 Deletion and Galactose Induction of GAL1 Activity—The plasmid p414GAL-CTR1myc was transformed into the strains ZY60 (WT), ZY89 (aft1Δ), and ZY90 (fet3Δ). Cells were grown in SC-TRP containing raffinose (2%), and Ctr1-Myc synthesis was induced by 0.5% galactose. Cells were treated with BPS and (NH4)Fe(SO4)2. Ctr1-Myc was detected as previously described. GAL promoter-driven SWE1 expression in strains SH26 and SH26aft1Δ was also analyzed with anti-Swe1 antibody (generously provide by Doug Kellog).
Growth Analysis of Iron-deficient Cells on Galactose Medium— Strains BY4741, BY4741aft1Δ, BY4741fet3Δ, and BY4741gal7Δ were streaked onto SC medium containing glucose or galactose. The plates were produced with or without (NH4)Fe(SO4)2. Cells were incubated at 30 °C for 4 days and photographed.
Galactose Induction of GAL Gene Expression Is Inhibited under Iron-deficient Conditions—We initially sought to find if changes in iron conditions affect copper uptake. Because Ctr1p undergoes Mac1p-dependent turnover in response to increasing copper concentrations (
), we used yeast strain ZY60, which does not express Mac1p. This strain allowed us to eliminate copper-triggered Ctr1p degradation. ZY60 strain with the p414GAL-CTR1myc tag was grown in standard laboratory medium (an iron-normal condition) and in medium containing the iron chelator, BPS, to cause iron deficiency (
). GAL1-driven Ctr1-Myc expression was induced by growth on galactose and analyzed by Western blotting using anti-Myc antibody (9E10). As shown in Fig. 1A, left panel, the Ctr1-Myc level was much lower in the BPS-treated samples than in the controls. Under the same conditions, no changes in phosphoglycerokinase (PGK) level were detected, indicating that the BPS effect was specific to CTR1 expression. Therefore, GAL1-driven CTR1 expression appears to be inhibited when iron is limited.
We were concerned that the observed effect of BPS might be caused by changes other than iron limitation. We reasoned that iron addition should ameliorate the change in CTR1 expression. To find if we were correct, we first treated cells with BPS and then with Fe3+ salt. The data in Fig. 1A, right panel, show that the addition of exogenous Fe3+ ions to the BPS-treated cells raised the Ctr1-Myc level in a dose-dependent fashion. We also compared GAL promoter-driven CTR1 expression (from p414GAL-CTR1myc) in the wild type (WT) and fet3Δ or aft1Δ mutants (
). Thus, these mutants would be expected to be relatively iron-starved compared with the wild type. The data in Fig. 1B show that the Ctr1-Myc levels were much lower in the two mutants than in the wild type. When the mutants were grown in Fe3+-supplemented medium the Ctr1-Myc levels increased significantly. In contrast, the PGK level remained constant under the same conditions. These results show that GAL1-driven CTR1 expression is indeed repressed under iron-deficient conditions.
To address a concern that GAL1 promoter activity might be inhibited by iron limitation, we repeated the above experiment using a single copy plasmid pCTR1myc (provided by Andy Dancis) in which CTR1-myc expression is under the control of the wild type CTR1 promoter (
). We initially detected a modest reduction in Ctr1-Myc level in BPS-treated cells with respect to control cells (data not shown). However, this result was not reproducible. These results led us to suspect that iron deficiency might instead inhibit galactose induction of the GAL1 promoter. To explore this possibility we used strain SH26, in which SWE1 is under the control of the GAL promoter and GAL-SWE1 is integrated into the genome. SWE1 encodes a protein kinase that is involved in the cell cycle (
), and there is currently no indication that SWE1 function is affected by iron. The cells were grown in galactose medium either in the absence or presence of BPS; Swe1p was detected by Western blotting with anti-Swe1 antibody. As presented in Fig. 1C, left panel, Swe1 level was dramatically reduced in the BPS-treated cells in comparison to that of the control cells. Furthermore, deletion of the chromosomal AFT1 gene in the SH26 strain background also resulted in a marked reduction in SWE1 expression (middle panel), which was corrected by Fe3+ supplementation (right panel). Based on these results, we concluded that the galactose induction of the GAL1 promoter is defective when iron is deficient.
Iron-deficient Mutants Show Defective Utilization of Galactose for Growth—The data in Fig. 1 indicate that the expression of galactose-metabolizing genes is likely reduced under iron-deficient conditions, so one could infer that the ability of iron-deficient cells to metabolize galactose would be reduced accordingly. We compared the growth of wild type (WT) and aft1Δ and fet3Δ cells on medium containing either glucose or galactose with or without Fe3+ salt. For controls we used an isogenic gal7Δ mutant. GAL7 encodes an uridyltransferase for metabolizing galactose (
). As shown in Fig. 2, left panel, the wild type, aft1Δ, fet3Δ, and gal7Δ strains grew equally well on glucose medium. On galactose medium, the aft1Δ and fet3Δ cells exhibited an obvious slow growth, the gal7Δ mutant did not grow at all, and the wild type grew normally (middle panel), indicating that the naturally iron-deficient aft1Δ and fet3Δ mutants are indeed defective in their ability to metabolize galactose. When high levels of exogenous Fe3+ ions were present, both the aft1Δ and fet3Δ mutants, but not the gal7Δ, were able to grow on galactose medium (right panel), suggesting that the defect of aft1Δ and fet3Δ cells to metabolize galactose is probably because of a lack of iron.
We have shown that galactose metabolism was altered by iron deficiency. We found that galactose-dependent induction of GAL promoter-driven gene expression was inhibited when iron was lacking and that iron-deficient cells had a diminished ability to grow on galactose medium. These two defects were corrected by iron replacement, demonstrating a requirement of iron for normal galactose metabolism. Therefore, iron homeostasis and galactose metabolism are connected with one other.
The physiology of galactose metabolism is well understood at the molecular level (for reviews, see Refs.
). The conversion is catalyzed by galactokinase, galactose-1-phosphate uridyltransferase, and uridine diphosphate galactose-4-epimerase. In the yeast S. cerevisiae, these enzymes are encoded by GAL1, GAL7, and GAL10, respectively. The expression of these GAL genes is galactose-inducible. The induction requires DNA binding by the transcriptional activator Gal4p. When glucose is present, the DNA binding of Gal4p is prevented by Gal80p, resulting in transcriptional repression of the GAL genes. Current thinking indicates that when cells are grown on galactose, an unidentified inducer molecule prevents Gal80p from inhibiting Gal4p function, thereby allowing expression of GAL genes. The inducer molecule has not been identified, but it was once believed to be a galactose metabolite that interacts with Gal3p (
How does iron deficiency inhibit the galactose induction of GAL gene expression? Because the uridyltransferase Gal7p is a Zn2+ and Fe2+ metalloprotein and the metal ions are apparently required for the transferase activity (
), it is possible that the activity of this galactose-metabolizing enzyme is reduced under iron-deficient conditions. This possibility is consistent with our observation that iron application rescued the growth of naturally iron-deficient cells on galactose medium (Fig. 2). However, if this were true, how could reduction in Gal7p activity inhibit GAL gene induction by galactose? One has to assume that galactose induction of the GAL gene is dependent on galactose metabolism itself as previously hypothesized (
) and that Gal7p might be involved in the production of an inducer molecule, perhaps UDP-Gal. We made this assumption because Gal7p catalyzes the production of UDP-Gal in the galactose metabolism pathway (
). To examine this assumption, we induced GAL-driven CTR1-MYC expression with galactose in the wild type and gal7Δ strains and treated cells with BPS. We found that CTR1-MYC expression was drastically reduced in the gal7Δ strain in comparison to the wild type (Fig. 3). This result offers further evidence that galactose induction of GAL gene expression is dependent on galactose metabolism. Furthermore, in the gal7Δ strain the CTR1-MYC expression was still repressed by BPS treatment as in the wild type, suggesting that a lack of iron might affect factor(s) other than Gal7p in terms of GAL gene expression. We also tested whether UDP-Gal is able to induce the GAL expression in the absence of galactose in intact cells and spheroplasts and found that GAL-driven CTR1-MYC expression was not induced (data not shown).
Additionally, an earlier study showed that galactose induction of GAL gene expression was affected by mitochondria function with respect to the IMP1 gene, which encodes an inner membrane peptidase (
). The wild type IMP1 strain could grow on and ferment galactose only in respiratory-deficient conditions. The strains carrying recessive allele imp1 of the IMP1 could also grow on and ferment galactose but only under respiratory-sufficient conditions (
), the iron deficiency-related inhibition of GAL gene induction could be because of defective mitochondria function. However, the strains used in this study all carry IMP1. When iron is lacking, one would expect the GAL gene to be induced by galactose and the naturally iron-deficient cells to be able to grow on galactose medium, which is not what we observed. Therefore, the effect of iron deficiency on GAL gene induction might not be wholly linked to mitochondria dysfunction. A recent genome-wide microarray that compared glucose- and galactose-grown wild type and GAL gene deletion mutants found that expression of iron and copper transporter genes was altered (
). This profile suggests that there might be coordination between sugar metabolism and metal ion homeostasis, at least with respect to iron and copper. Also, iron might influence galactose metabolism through a mechanism that has yet to be discovered.
The discovery that iron deficiency impairs the ability of a cell to metabolize galactose might have direct clinical applications. A study reported (
) that even in the United States, 3% of toddlers and 2–5% of teenage girls are sufficiently iron-deficient to develop anemia. Those individuals who lack sufficient iron might also suffer a secondary defect in galactose metabolism. The finding might also help to explain why an infant needs more iron than an adult to support his rapid growth (
), because galactose is formed by the hydrolysis of lactose-rich breast milk. Because copper deficiency can result in iron deficiency, copper homeostasis might indirectly influence galactose metabolism as well. Future studies are warranted to determine the molecular mechanisms that link metal ions to galactose metabolism. This work has provided an opening into the exploration of the impact of metal ion homeostasis on the genetic foundations of sugar metabolism.
A clear implication of this fortuitous finding concerns the use of GAL promoters to direct gene expression in studying iron homeostasis. Because GAL gene promoters show high levels of inducibility, they are widely used to direct gene expression (
). In light of the data reported here, the use of GAL-driven expression systems to investigate Fe homeostasis should be carefully controlled and interpreted with caution.
We thank Drs. Andrew Dancis for the AFT1 deletion plasmid and David Eide for FET3 disruption plasmid. We are grateful to Drs. Dennis Winge, Grant Hartzog, Doug Kellogg, and Chris Vulpe for providing yeast strains.