Relationship between UDP-Galactose 4′-Epimerase Activity and Galactose Sensitivity in Yeast
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m600778200
ISSN1083-351X
AutoresJamie Wasilenko, Judith L. Fridovich‐Keil,
Tópico(s)Polyamine Metabolism and Applications
ResumoUDP-galactose 4′-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to ∼5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to ∼5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems. UDP-galactose 4′-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to ∼5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to ∼5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems. Galactose is metabolized in species ranging from Escherichia coli to mammals via a series of reactions collectively known as the Leloir pathway. The three enzymes that catalyze these sequential reactions are galactokinase (EC 2.7.1.6), galactose-1-phosphate uridylyltransferase (GALT 2The abbreviations used are: GALT, galactose-1-phosphate uridylyltransferase; GALE, UDP-galactose 4′-epimerase; gal-1P, galactose 1-phosphate. , EC 2.7.7.12), and UDP-galactose 4′-epimerase (GALE, EC 5.1.3.2) (see Fig. 1 (1Holden H.M. Rayment I. Thoden J.B. J. Biol. Chem. 2003; 278: 43885-43888Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)). Deficiency of any one of these enzymes in humans results in a form of the inherited metabolic disorder, galactosemia (2Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Eds. A. Childs B. Kinzler K.W. Vogelstein B. Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Book Co., New York2000: 1553-1587Google Scholar). The most common and clinically severe form of galactosemia is classic galactosemia (OMIM entry 230400), which affects about 1 in 30,000–60,000 live births and results from profound impairment of the GALT enzyme. Although typically asymptomatic at birth, patients with classic galactosemia develop escalating symptoms after exposure to a milk-based diet. In the absence of intervention these symptoms, which include vomiting, diarrhea, cataracts, hepatomegaly, and E. coli sepsis, can be lethal within the first few days to weeks of life. Although a dietary restriction of galactose, the current standard of care, relieves or prevents the acute and potentially lethal symptoms, many patients with classic galactosemia go on to develop long term complications. The most common complications include speech and/or learning disabilities in 30–50% of all patients and primary or premature ovarian failure in almost 85% of females (3Waggoner D.D. Buist N.R.M. Donnell G.N. J. Inherited Metab. Dis. 1990; 13: 802-818Crossref PubMed Scopus (463) Google Scholar). Perhaps the least well understood form of galactosemia is epimerase (GALE) deficiency galactosemia (OMIM entry 230350). As illustrated in Fig. 1, human GALE catalyzes not only the interconversion of UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) but also the intercon-version of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine (e.g. Refs 4Piller F. Hanlon M.H. Hill R.L. J. Biol. Chem. 1983; 258: 10774-10778Abstract Full Text PDF PubMed Google Scholar and 5Schulz J. Watson A. Sanders R. Ross K. Thoden J. Holden H. Fridovich-Keil J. J. Biol. Chem. 2004; 279: 32796-32803Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Originally described as a "peripheral" clinically benign condition in which GALE deficiency is restricted to the circulating red and white blood cells (6Gitzelmann R. Helv. Paediat. Acta. 1972; 27: 125-130PubMed Google Scholar, 7Gitzelmann R. Steimann B. Helv. Paediat. Acta. 1973; 28: 497-510PubMed Google Scholar, 8Gitzelmann R. Steinmann B. Mtchell B. Haigis E. Helv. Paediat. Acta. 1976; 31: 441-452Google Scholar), GALE deficiency was later demonstrated to exist also in an extremely rare but clinically severe "generalized" form characterized by enzyme impairment in multiple tissues and symptoms reminiscent of classic galactosemia (9Holton J.B. Gillett M.G. MacFaul R. Young R. Arch. Dis. Child. 1981; 56: 885-887Crossref PubMed Scopus (84) Google Scholar, 10Sardharwalla I.B. Wraith J.E. Bridge C. Fowler B. Roberts S.A. J. Inherited Metab. Dis. 1988; 11: 249-251Crossref PubMed Scopus (40) Google Scholar, 11Walter J.H. Roberts R.E.P. Besley G.T.N. Wraith J.E. Cleary M.A. Holton J.B. MacFaul R. Arch. Dis. Child. 1999; 80: 374-376Crossref PubMed Scopus (67) Google Scholar). Most recently, GALE deficiency has been described as a continuous disorder, with a spectrum of enzyme impairment and corresponding metabolic compromise impacting a variety of tissues in affected individuals (12Schulpis K.H. Michelakakis H. Charokopos E. Papakonstantinou E. Messaritakis J. Shin Y. J. Inherited Metab. Dis. 1993; 16: 1059-1060Crossref PubMed Scopus (4) Google Scholar, 13Schulpis K.H. Papakonstantinou E.D. Koidou A. Michelakakis H. Tzamouranis J. Patsouras A. Shin Y. J. Inherited Metab. Dis. 1993; 16: 903-904Crossref PubMed Scopus (6) Google Scholar, 14Quimby B.B. Alano A. Almashanu S. DeSandro A.M. Cowan T.M. Fridovich-Keil J.L. Am. J. Hum. Gen. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 15Alano A. Almashanu S. Chinsky J.M. Costeas P. Blitzer M.G. Wulfsberg E.A. Cowan T.M. J. Inherited Metab. Dis. 1998; 21: 341-350Crossref PubMed Scopus (49) Google Scholar, 16Shin Y.S. Korenke G.C. Huddke P. Knerr I. Podskarbi T. J. Inherit. Metab. Dis. 2000; 23: 383-386Crossref PubMed Scopus (11) Google Scholar, 17Openo K. Schulz J. Vargas C. Orton C. Epstein M. Schnur R. Scaglia F. Berry G. Gottesman G. Ficicioglu C. Slonim A. Shroer R. Yu C. Rangel V. Kenan J. Lamance K. Fridovich-Keil J. Am. J. Hum. Genet. 2006; 78: 89-102Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Despite decades of study, the underlying bases of pathophysiology in both classic and epimerase-deficiency galactosemia remain unknown (2Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Eds. A. Childs B. Kinzler K.W. Vogelstein B. Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Book Co., New York2000: 1553-1587Google Scholar). For example, although it is clear that patients with both disorders accumulate high levels of galactose 1-phosphate (gal-1P) on galactose-containing diets, whether gal-1P causes pathophysiology or simply correlates with pathophysiology remains unclear. In the absence of an animal model of galactosemia that recapitulates the human phenotype (18Leslie N.D. Yager K.L. McNamara P.D. Segal S. Biochem. Mol. Med. 1996; 59: 7-12Crossref PubMed Scopus (78) Google Scholar, 19Leslie N. Bai S. Mol. Genet. Metab. 2001; 72: 31-38Crossref PubMed Scopus (6) Google Scholar, 20Ning C. Reynolds R. Chen J. Yager C. Berry G.T. McNamara P.D. Leslie N. Segal S. Pediatr. Res. 2000; 48: 211-217Crossref PubMed Scopus (52) Google Scholar, 21Ning C. Reynolds R. Chen J. Yager C. Berry G.T. Leslie N. Segal S. Mol. Genet. Metab. 2001; 72: 306-315Crossref PubMed Scopus (34) Google Scholar), we and others have turned to the single celled yeast, Saccharomyces cerevisiae, as a biochemically and genetically amenable model to explore the metabolic and cellular consequences of impaired GALT and GALE function in eukaryotes. Studies dating back more than 40 years (22Douglas H.C. Hawthorne D.C. Genetics. 1964; 49: 837-844Crossref PubMed Google Scholar) demonstrate that both GALT and GALE-null S. cerevisiae arrest their growth in response to even trace quantities (0.05%) of environmental galactose despite the presence of an alternate, metabolize-able carbon source (e.g. glycerol/ethanol). More recent studies by our group and others (23Mehta D.V. Kabir A. Bhat P.J. Biochim. Biophys. Acta. 1999; 1454: 217-226Crossref PubMed Scopus (38) Google Scholar, 24Kabir M.A. Khanday F.A. Mehta D.V. Bhat P.J. Mol. Gen. Genet. 2000; 262: 1113-1122Crossref PubMed Scopus (15) Google Scholar, 25Lai K. Elsas L. Biochem. Biophys. Res. Commun. 2000; 271: 392-400Crossref PubMed Scopus (59) Google Scholar, 26Wohlers T. Fridovich-Keil J.L. J. Inherited Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 27Riehman K. Crews C. Fridovich-Keil J.L. J. Biol. Chem. 2001; 276: 10634-10640Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar) have both confirmed and extended that result. For example, we found that GALE-null yeast arrest growth at galactose exposures 10-fold lower than do GALT-null yeast, although the GALT-null yeast accumulate levels of gal-1P greater than or equal to their GALE-null counterparts (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). We also found that although GALT-null yeast remain at least marginally competent to deplete their medium of galactose, GALE-null yeast cannot do so, even after prolonged incubation (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Of note, this same observation was corroborated recently in mammalian and patient cells (17Openo K. Schulz J. Vargas C. Orton C. Epstein M. Schnur R. Scaglia F. Berry G. Gottesman G. Ficicioglu C. Slonim A. Shroer R. Yu C. Rangel V. Kenan J. Lamance K. Fridovich-Keil J. Am. J. Hum. Genet. 2006; 78: 89-102Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 29Schulz J. Ross K. Malmstrom K. Krieger M. Fridovich-Keil J. J. Biol. Chem. 2005; 280: 13493-13502Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). These data raise the possibility that although the acute phenotypes of profound GALT or GALE impairment may be similar in patients and in yeast, the mechanism(s) of pathophysiology of galactose sensitivity for GALT-versus GALE-impaired cells may be distinct. Previously we addressed the quantitative relationship between GALT impairment and galactose sensitivity by expressing each of 16 differentially impaired patient alleles of human GALT in a null-background strain of yeast (27Riehman K. Crews C. Fridovich-Keil J.L. J. Biol. Chem. 2001; 276: 10634-10640Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and defining the galactose sensitivity of the resulting strains. Our results demonstrated a clear inverse relationship between the level of residual GALT activity and sensitivity of the corresponding strains to galactose. Furthermore, it appeared that human GALT alleles associated with greater than about 10% wild-type human GALT activity, corresponding to about 3% yeast GAL7 activity, were indistinguishable from wild-type cells. Here we have addressed the quantitative relationship between GALE impairment and galactose metabolism and sensitivity in yeast. Although conceptually parallel to our earlier work, the results of these studies demonstrate both important similarities and important contrasts to GALT. In particular, we have asked three questions. 1) Is GALE rate-limiting for galactose metabolism in otherwise Leloir wild-type yeast and, if so, over what range? 2) What is the relationship between GALE activity and galactose-dependent growth impairment in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? With this last question we are seeking to know if some galactose metabolites may correlate more closely with the degree of galactose sensitivity than do others, perhaps implicating a role for these compounds in the mechanism of sensitivity. To ask these questions we inserted a doxycycline-regulated promoter just upstream of the GAL10 open reading frame in otherwise Leloir wild-type yeast, rendering the encoded GALE enzyme doxycycline repressible. We then grew these cells under conditions of differential drug concentration and followed their growth and metabolic response to galactose exposure. GAL10 wild-type and gal10-null yeast served as positive and negative controls, respectively. Our results demonstrated that the answer to the first question was, yes. There was a clear linear relationship between residual GALE activity and galactose metabolism in otherwise Leloir wild-type yeast over a range from 0 to about 5% wild-type GALE. Above about 5% GALE was no longer limiting. The answer to the second question was that although all of the yeast grew similarly in the absence of galactose, in the presence of galactose there was a clear step relationship between growth rate and GALE activity, with a threshold for growth at about 5% wild-type GALE. Finally, in answer to the third question, GALE showed an inverse, near linear relationship with accumulation of abnormal levels of gal-1P and UDP-gal, again up to a threshold of about 5% wild-type GALE. Above 6% GALE appeared to be in excess for all of the outcomes measured. Together, these data both quantify the role of GALE and define key similarities and distinctions between GALT and GALE as mediators of galactose metabolism and sensitivity in yeast. Yeast Strains and Manipulation—All yeast manipulations were performed according to standard protocols (30Guthrie C. Fink G. Methods Enzymol.194. 1991: 1-863Google Scholar) using haploid strains derived from W303 (MATa ade2–1 his3–11,15 leu 2–3,112 ura3–1 trp1–1 can1–100 RAD5+), which was the kind gift of Dr. Rodney Rothstein, Columbia University, New York, NY. To enable the expression of endogenous yeast GAL genes in both the presence and absence of galactose, all strains were rendered gal80-null by one-step gene replacement, as described previously (31Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (752) Google Scholar). The negative control strain JFy4931 was also rendered gal10-null by the same method (31Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (752) Google Scholar). The dox.GAL10 yeast strain (JFy4828) was constructed by insertion of a cassette containing a doxycycline-repressible promoter (32Belli G. Gari E. Aldea M. Herrero E. Yeast. 1998; 14: 1127-1138Crossref PubMed Scopus (132) Google Scholar, 33Belli G. Gari E. Piedrafita L. Aldea M. Herrero E. Nucleic Acids Res. 1998; 26: 942-947Crossref PubMed Scopus (227) Google Scholar) just upstream of the endogenous GAL10 open reading frame. This manipulation was achieved by one-step gene replacement using a PCR-generated fragment (from plasmid CM225) amplified to carry flanking sequences homologous to the desired insertion site. To tighten regulatory control over the doxycycline-repressible promoter, a second cassette encoding a repressor moiety (from plasmid CM244) was also integrated into the LEU2 locus, as described elsewhere (32Belli G. Gari E. Aldea M. Herrero E. Yeast. 1998; 14: 1127-1138Crossref PubMed Scopus (132) Google Scholar, 33Belli G. Gari E. Piedrafita L. Aldea M. Herrero E. Nucleic Acids Res. 1998; 26: 942-947Crossref PubMed Scopus (227) Google Scholar). All integrations were confirmed by genomic PCR of the resulting strains. Yeast Culture Conditions—All yeast were cultured at 30 °C in yeastrich growth medium containing 2% glycerol, 2% ethanol (YPGE) unless otherwise indicated, as in Fig. 3 and Supplemental Fig. 2E, for which yeast were cultured in synthetic medium containing 2% glycerol, 2% ethanol (SGE). Cells were exposed to doxycycline as follows. On day 1, a given colony was inoculated into medium containing 3 μg/ml doxycycline to completely shut down expression of the dox.GAL10 allele. After growth overnight cells were harvested by centrifugation, washed 3 times with sterile water to remove residual drug, and resuspended to an OD600 ∼ 0.05 in medium containing the desired final concentration of doxycycline (e.g. 0–80 ng/ml). Cells were grown in this medium to an OD600 of ∼1.5, at which point they were diluted into fresh medium containing the same level of drug and allowed to grow for another approximately eight doublings under these conditions. Relevant doxycycline concentrations are indicated for each experiment. Leloir Enzyme Activity Assays from Soluble Yeast Lysates—Soluble yeast protein lysates were prepared as follows. Cell pellets were washed with water and resuspended in lysis buffer (20 mm Hepes/KOH (pH 7.5), 1 mm dithiothreitol, and 0.3 mg bovine serum albumin/ml) supplemented with protease inhibitors (complete mini protease inhibitor mixture, Roche Applied Science). Lysis was carried out by vigorous agitation with 0.5-mm acid-washed glass beads at 4 °C. Lysates were clarified by centrifugation at high speed in an Eppendorf microcentrifuge for 10 min at 4 °C. To remove small metabolites, supernatants were passed through Bio-Spin 30 columns (Bio-Rad) before protein quantification. Protein concentration was then determined using the Bio-Rad protein reagent, as recommended by the manufacturer, with bovine serum albumin as the standard. Samples were stored at –85 °C until use. Galactokinase activity was evaluated in samples by measuring the conversion of galactose to gal-1P, essentially as described previously (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Assays were stopped by the addition of 450 μl of ice-cold water followed immediately by filtration through 0.2-μm nylon micro-spin columns (Corning) to remove proteins and particulates before high performance liquid chromatography analysis. GALT activity was evaluated by measuring the conversion of gal-1P into glucose 1-phosphate, essentially as described previously (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Assays were stopped by the addition of 450 μl of ice-cold water followed immediately by filtration as described above. GALE activity was evaluated in samples by monitoring the conversion of UDP-gal to UDP-glc, essentially as described previously (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Assays were stopped by the addition of 237.5 μl of ice-cold water followed immediately by filtration as described above. Yeast Growth Studies—Yeast cultures were monitored for growth in liquid medium as described previously (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). In brief, mid-log phase cells were diluted into fresh medium as indicated in the wells of a 96-well plate (NUNC), which was then maintained at 30 °C with constant agitation. OD600 measurements were recorded every 2 h using a microplate reader (Bio-Tek Instruments). Metabolite Analysis—Both consumption of external galactose and abundance of intracellular metabolites were measured as described previously (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Doxycycline-regulated Expression of GALE in Yeast—To explore the quantitative relationship between GALE activity and galactose metabolism and sensitivity in yeast, we generated a strain in which the expression of GAL10, encoding GALE, was regulated by doxycycline. In brief, we introduced a doxycycline-repressible promoter (32Belli G. Gari E. Aldea M. Herrero E. Yeast. 1998; 14: 1127-1138Crossref PubMed Scopus (132) Google Scholar, 33Belli G. Gari E. Piedrafita L. Aldea M. Herrero E. Nucleic Acids Res. 1998; 26: 942-947Crossref PubMed Scopus (227) Google Scholar) just upstream of the GAL10 coding sequence in a strain (JFy4763) that was otherwise wild type for all Leloir pathway enzymes and that was engineered to express the appropriate doxycycline-responsive transcriptional activator and repressor protein moieties. This strain was also gal80-null, enabling strong expression of the endogenous GAL genes despite the absence of galactose (34Pilauri V. Bewley M. Diep C. Hopper J. Genetics. 2005; 169: 1903-1914Crossref PubMed Scopus (46) Google Scholar). The resulting dox.GAL10 strain of yeast was designated JFy4828. To confirm both the expression and regulation of GALE in JFy4828 as well as to ensure that galactokinase and GALT expression remained intact, we assessed all three Leloir enzyme activities in both the dox. GAL10 and parental strains cultured in both the presence and absence of 3 μg/ml doxycycline. Our results demonstrated that the levels of galactokinase and GALT activity in both the parental and dox.GAL10 strains were statistically indistinguishable regardless of the presence or absence of drug (Table 1). Similarly, GALE activity in the parental strain was unaffected by the presence or absence of drug. In contrast, GALE in the dox.GAL10 strain was expressed at about 26% that of the control level in the absence of drug and was undetectable in the presence of drug (Table 1). This ∼4-fold drug-independent drop of GALE activity in the dox.GAL10 strain relative to the wild-type strain presumably reflects the weaker basal strength of the doxycycline-regulated promoter compared with the endogenous GAL10 promoter.TABLE 1Galactokinase, GALT, and GALE enzyme activities in wild-type and dox.GAL10 yeast cultured in the presence and absence of doxycycline Values are the mean ± S.E., n = 3, except where indicated.EnzymeTreatmentWild-type straindox.GAL10 strainμmol/mg prot/minμmol/mg protein/minGalactokinaseNo drug2.66 ± 0.122.39; 2.34 (n = 2)3 μg/ml doxycycline1.49 ± 0.522.96 ± 0.17GALTNo drug1.60 ± 0.181.82 ± 0.493 μg/ml doxycycline1.25 ± 0.281.89 ± 0.16GALENo drug1.40 ± 0.110.358 ± 0.033 μg/ml doxycycline1.56 ± 0.040 ± 0.00aStatistical significance.a Statistical significance. Open table in a new tab As a test of the impact of regulated GALE expression in the dox.GAL10 strain, we followed the growth of wild-type, dox.GAL10, and gal10-null yeast cultured in the presence versus absence of 3 μg/ml doxycycline and also in the presence versus absence of 0.025% galactose (Fig. 2). We have previously demonstrated that although wild-type yeast grow well in SGE medium containing glycerol/ethanol as the carbon source regardless of the presence or absence of galactose, gal10-null yeast grow well in the absence of galactose but arrest growth completely in the presence of as little as 0.002% galactose (26Wohlers T. Fridovich-Keil J.L. J. Inherited Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Consistent with that result, we observed here that in the absence of doxycycline (Fig. 2, left panel), both wild-type and dox.GAL10 yeast grew well in both the absence and presence of galactose. In the presence of 3 μg/ml doxycycline (Fig. 2, right panel), however, only the wild-type strain grew well in both the presence and absence of galactose; both the dox.GAL10 and gal10-null yeast completely arrested growth upon the addition of galactose to the medium. Together, these data corroborate the in vitro GALE activity assay results described above and further demonstrate that 26% wild-type GALE activity is sufficient to confer galactose resistance in yeast. Loss of Epimerase Activity, Not Mutarotase Activity, Mediates Galactose Sensitivity of gal10-null Yeast—Unlike human GALE, the protein product of the yeast GAL10 gene, Gal10p, is a fusion protein that exhibits both UDP-galactose 4′-epimerase activity and also galactose mutarotase activity (35Majumdar S. Ghatak J. Mukherji S. Bhattacharjee H. Bhaduri A. Eur. J. Biochem. 2004; 271: 753-759Crossref PubMed Scopus (45) Google Scholar, 36Thoden J.B. Holden H.M. J. Biol. Chem. 2005; 280: 21900-21907Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Deletion or repression of GAL10, therefore, results in the loss of both activities. To test whether the galactose-dependent growth arrest observed in gal10-null yeast reflected loss of epimerase, mutarotase, or both activities, we tested the galactose sensitivity of gal10-null yeast engineered to express human GALE, which complements the loss of epimerase activity but not the loss of mutarotase activity. As illustrated in Fig. 3, human GALE fully complemented the growth arrest of gal10-null yeast exposed to galactose, thereby demonstrating that it is loss of epimerase and not loss of mutarotase activity that underlies the galactose sensitivity of gal10-null yeast. Titration of GALE Activity in dox.GAL10 Yeast—To investigate the quantitative relationship between GALE and galactose sensitivity in yeast, we first defined the relationship between doxycycline exposure and GALE activity in our dox.GAL10 strain. As described under "Experimental Procedures," initial cultures were exposed to 3 μg/ml doxycycline to completely repress GALE expression, after which point cells were diluted into fresh medium containing the desired level of drug (0–80 ng/ml). After ∼40 h of continuous culture at the desired level of doxycycline, at which point GALE activity levels had stabilized, yeast were harvested and assayed for GALE activity relative to a wild-type control. As illustrated in Fig. 4, there was a clear and reproducible inverse relationship between the level of drug exposure and the level of GALE activity in the dox.GAL10 yeast. In particular, cells cultured in the absence of drug demonstrated 26% GALE activity, cells cultured in the presence of 25 ng/ml demonstrated about 12% GALE, cells cultured in the presence of 35–50 ng/ml drug demonstrated 55 ng/ml drug demonstrated 6% GALE activity were only marginally impaired, if at all. Indeed, by the 16-h time point there appeared to be a near-linear relationship (r2 = 0.89) between the percentage of external galactose remaining in the culture medium and GALE activity, up to a threshold of ∼6% wild-type GALE, above which all cultures fully depleted their medium of galactose. Internal metabolites followed a similar pattern, such that elevated levels of intracellular galactose, gal-1P, and UDP-gal also demonstrated a near-linear inverse relationship with GALE activity (r2 = 0.70, 0.79, and 0.55, respectively), up to a threshold of about 5–6% wild-type GALE, above which point metabolites essentially normalized. Finally, abnormal depletion of UDP-glc in the presence of galactose was reproducibly seen only at the very lowest levels of GALE activity (e.g. gal10-null strain and dox.GAL10 strain in the presence of drug). We have addressed three fundamental questions regarding the role of GALE in galactose metabolism and sensitivity in yeast. We have asked the following. 1) Is GALE rate-limiting for galactose metabolism in otherwise Leloir-wild-type yeast and, if so, over what range of activity? 2) What is the relationship between GALE activity and yeast growth rate in the presence of galactose? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? In answering these questions we have both delineated the role of GALE in galactose sensitivity in yeast and also contrasted this role with that of GALT. In addition, we have further set the stage for future studies in mammalian cells aimed at exploring the role of GALE as a mediator of pathophysiology and patient outcome in patients with galactosemia. Role of GALE in Galactose Metabolism in Yeast—We found that the ability of yeast to deplete their medium of galactose was rate-limited by GALE over a range from 0–5% wild-type activity, and this relationship was remarkably linear (r2 = 0.89). Yeast expressing GALE activity levels above 6% depleted environmental galactose as quickly and completely as did their wild-type counterparts. Although the precise threshold is likely a function of the amount of galactose added, among other factors, the important conclusion is that, like GALT (27Riehman K. Crews C. Fridovich-Keil J.L. J. Biol. Chem. 2001; 276: 10634-10640Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), GALE is expressed in marked excess of Leloir requirements in normal yeast. Relationship between GALE Activity and Growth Impairment in Yeast—A comparison of strains cultured in YPGE medium spiked with 0.025% galactose and different levels of doxycycline demonstrated an inverse step function relationship between growth rate and GALE activity. At less than 4% GALE, growth rates were almost 0; between 4 and 5% GALE, cultures demonstrated a long lag, after which some growth became evident; above 6% GALE, growth rates were normal. It is interesting to note that the lag in growth of the 4–5% GALE cultures corresponded precisely with the time frame of external galactose depletion for these cells. These data suggest that whereas galactose metabolism may be a linear function of GALE activity within a limiting range (0 to 5%), the growth response of yeast to that metabolic block is not linear. These data are strikingly similar, at least in qualitative terms, to our earlier data with GALT (27Riehman K. Crews C. Fridovich-Keil J.L. J. Biol. Chem. 2001; 276: 10634-10640Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In particular, we previously observed that yeast expressing <1% human GALT activity, corresponding to <0.3% yeast GALT activity, could not grow in the presence of galactose, whereas yeast expressing <5% human GALT, corresponding to 10% human GALT activity, corresponding to >3% yeast GALT activity, grew indistinguishably from the wild-type control. Especially considering that the level of galactose used in the GALT experiments (0.05%) was twice the level used here (0.025%), these data suggest that whereas the patterns may be qualitatively similar, yeast are quantitatively hypersensitive to the loss of GALE relative to the loss of GALT. These data are fully consistent with our earlier results demonstrating that GALE-null yeast growth arrest upon exposure to galactose levels 10-fold lower than do their GALT-null counterparts (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). One important factor that may contribute to this apparent discrepancy between GALT and GALE requirements is that whereas GALE is unique in its ability to interconvert UDP-gal and UDP-glc, GALT is not unique in its ability to "convert" gal-1P into UDP-gal. The enzyme UDP-glucose pyrophosphorylase (UGP1) provides an inefficient albeit effective alternate route for this reaction (23Mehta D.V. Kabir A. Bhat P.J. Biochim. Biophys. Acta. 1999; 1454: 217-226Crossref PubMed Scopus (38) Google Scholar, 25Lai K. Elsas L. Biochem. Biophys. Res. Commun. 2000; 271: 392-400Crossref PubMed Scopus (59) Google Scholar, 37Leslie N. Yager C. Reynolds R. Segal S. Mol. Genet. Metab. 2005; 85: 21-27Crossref PubMed Scopus (12) Google Scholar). Cells may, therefore, be able to manage with less GALT activity because UGP1 carries part of the load. Relationship between GALE Activity and Abnormal Galactose Metabolite Levels—One obvious result of impaired GALE activity in yeast exposed to environmental galactose is the accumulation of abnormal levels of intracellular galactose metabolites, including galactose, gal-1P, and UDP-gal (28Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Genet. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). We explored the possibility that one or more of these metabolites might correlate with the step-function growth restriction of GALE-impaired yeast but found that within the key range of 0–5% enzyme activity, each measured metabolite demonstrated a clear linear inverse relationship with GALE. Furthermore, only yeast with very low GALE activity levels (<1%) showed a decreased abundance of UDP-glc in the presence of galactose. These data suggest that if abnormal accumulation of the metabolites measured indeed underlies the galactose-dependent growth restriction observed, either the impact of individual metabolites must be synergistic, producing an apparent step function in growth, or else the sensitivity must follow a steep threshold such that even the slightest accumulation of gal-1P or UDP-gal results in growth arrest. Future experiments that manipulate the accumulation of individual metabolites independently will be required to distinguish between these possibilities. We gratefully acknowledge the many helpful contributions of other members of the Fridovich-Keil laboratory, in particular Dr. Kerry Ross. Download .pdf (.54 MB) Help with pdf files
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