Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase
2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês
10.1074/jbc.m009583200
ISSN1083-351X
AutoresKristen Riehman, Charity Crews, Judith L. Fridovich‐Keil,
Tópico(s)Folate and B Vitamins Research
ResumoImpairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans. Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans. galactose-1-phosphate uridylyltransferase human GALT galactose 1-phosphate The enzyme galactose-1-phosphate uridylyltransferase (GALT)1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1Frey P.A. FASEB J. 1996; 10: 461-470Crossref PubMed Scopus (379) Google Scholar, 2Holton, J. B., Walter, J. H., Tyfield, L. A. (2000) inMetabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, S. W., Valle, D., Eds., A., Childs, B., Kinzler, K. W., and Vogelstein, B., Assoc. eds) McGraw-Hill Inc., New York, pp. 1553–1587.Google Scholar). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2Holton, J. B., Walter, J. H., Tyfield, L. A. (2000) inMetabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, S. W., Valle, D., Eds., A., Childs, B., Kinzler, K. W., and Vogelstein, B., Assoc. eds) McGraw-Hill Inc., New York, pp. 1553–1587.Google Scholar,3Segal S. Berry G. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York1995: 967-1000Google Scholar). Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30–50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4Waggoner D.D. Buist N.R.M. Donnell G.N. J. Inherited Metab. Dis. 1990; 13: 802-818Crossref PubMed Scopus (455) Google Scholar). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2Holton, J. B., Walter, J. H., Tyfield, L. A. (2000) inMetabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, S. W., Valle, D., Eds., A., Childs, B., Kinzler, K. W., and Vogelstein, B., Assoc. eds) McGraw-Hill Inc., New York, pp. 1553–1587.Google Scholar and 3Segal S. Berry G. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York1995: 967-1000Google Scholar), the biochemical mechanism of pathophysiology in galactosemia remains unknown. One of the fundamental questions with regard to classic galactosemia concerns the identification of predictive factors that might be used to distinguish those patients who will thrive long term from those who will experience complications. Waggoner et al. (4Waggoner D.D. Buist N.R.M. Donnell G.N. J. Inherited Metab. Dis. 1990; 13: 802-818Crossref PubMed Scopus (455) Google Scholar) addressed this issue a decade ago with an international retrospective questionnaire study and found no clear correlation between long term outcome and three of the most obvious candidate extrinsic factors: age at diagnosis, presence of neonatal complications before treatment, or strict dietary compliance. These data suggested that some intrinsic factor(s) might serve a predominant role in defining outcome. With regard to intrinsic factors, perhaps the most obvious is GALT genotype. The number of candidate mutations identified in patient alleles now exceeds 150, the majority of which are missense point mutations (5Tyfield L. Reichardt J. Fridovich-Keil J. Croke D.T. Elsas L.J. Strobl W. Kozak L. Coskun T. Novelli G. Okano Y. Zekanowski C. Shion Y. Boleda M.D. Hum. Mutat. 1999; 13: 417-430Crossref PubMed Scopus (143) Google Scholar). Indeed many if not most galactosemia patients studied are compound heterozygotes, further complicating the picture. Although most naturally occurring mutant alleles of hGALT have not been well characterized with regard to function, high sensitivity biochemical studies of large groups of ungenotyped patients have demonstrated clear biochemical heterogeneity in the patient population (6Ng W.G. Xu Y.-K Kaufman F.R. Lee J.E.S. Donnell G.N. Neonatal Screening in the Nineties.in: Wilcken B. Webster D. 8th International Neonatal Screening Symposium and Inaugural Meeting of the International Society for Neonatal Screening. Leura, Australia1991: 181-188Google Scholar, 7Xu Y.-K Kaufman F.R. Donnell G.N. Ng W.G. Clin. Chim. Acta. 1995; 235: 125-136Crossref PubMed Scopus (13) Google Scholar). Furthermore, differences in the prevalence of specific hGALT genotypes also have been observed in populations of patients withversus without detectable GALT activity (8Wang B.B.T. Xu Y.-K Ng W.G. Wong L.-J.C. Mol. Genet. Metab. 1998; 63: 263-269Crossref PubMed Scopus (34) Google Scholar). These data support the hypothesis that genotype may correlate with activity, which in turn may influence metabolite levels and phenotypic outcome. Indeed, ungenotyped patients with detectable GALT activity have been reported to accumulate lower levels of gal-1-P and to experience a milder clinical course and than do their counterparts without detectable GALT activity (6Ng W.G. Xu Y.-K Kaufman F.R. Lee J.E.S. Donnell G.N. Neonatal Screening in the Nineties.in: Wilcken B. Webster D. 8th International Neonatal Screening Symposium and Inaugural Meeting of the International Society for Neonatal Screening. Leura, Australia1991: 181-188Google Scholar, 7Xu Y.-K Kaufman F.R. Donnell G.N. Ng W.G. Clin. Chim. Acta. 1995; 235: 125-136Crossref PubMed Scopus (13) Google Scholar). Similarly, a number of studies report decreased levels of UDP-gal and altered ratios of UDP-glc/UDP-gal in samples from galactosemic patients compared with controls (2Holton, J. B., Walter, J. H., Tyfield, L. A. (2000) inMetabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, S. W., Valle, D., Eds., A., Childs, B., Kinzler, K. W., and Vogelstein, B., Assoc. eds) McGraw-Hill Inc., New York, pp. 1553–1587.Google Scholar). Nonetheless, there has been no direct test of the relationship of these parameters. Furthermore, although some retrospective outcome studies of patients with galactosemia have reported a statistically significant relationship between genotype and outcome, others have not (9Elsas L.J. Langley S. Paulk E.M. Hjelm L.N. Dembure P.P. Eur. J. Pediatr. 1995; 154, (7 Suppl. 2): S21-S27Crossref Scopus (50) Google Scholar, 10Hirokawa H. Okano Y. Asada M. Fujimoto A. Suyama I. Isshiki G. Eur. J. Hum. Genet. 1999; 7: 757-764Crossref PubMed Scopus (21) Google Scholar, 11Kaufman F.R. Reichardt J.K. Ng W.G. Xu Y.K. Manis F.R. McBride-Chang C. Wolff J.A. J. Pediatr. 1994; 125: 225-227Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Shield J.P. Wadsworth E.J. MacDonald A. Stephenson A. Tyfield L. Holton J.B. Marlow N. Arch. Dis. Child. 2000; 83: 248-250Crossref PubMed Scopus (53) Google Scholar), perhaps reflecting the complications of confounding variables and limited sample sizes. We report here the first quantitative and allele-specific test of the hypothesis that there is a relationship between hGALT genotype, activity, metabolite levels, and sensitivity to galactose in a eukaryotic system. In particular, we have used yeast, extending from initial observations by Douglas and Hawthorne (13Douglas H.C. Hawthorne D.C. Genetics. 1964; 49: 837-844Crossref PubMed Google Scholar), who reported that GALT-deficient yeast, but not their wild-type counterparts, were growth-arrested by the addition of small amounts of galactose to the medium despite the presence of other metabolizable carbon sources. We applied a previously described null-background yeast expression system (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar) to study 16 naturally occurring patient alleles of human GALT (R67C, S135L, L139P, V151A, F171S, P183T, Q188R, R201H, R231H, R259W, K285N, E291K, N314D, R333W, Y323D, and T350A). In particular, we characterized each allele in terms of both abundance and activity of the encoded human GALT protein. Three main groups of alleles were identified: those with <1% wild-type activity, those with 1–5% wild-type activity, and those with ≥∼10% wild-type activity. Monitoring the impact of galactose exposure on strains of yeast expressing each mutant allele, we observed that, with few exceptions, yeast expressing the lowest activity hGALT proteins demonstrated the most significant sensitivity to galactose and the most prolonged accumulation of gal-1-P, an indicator of galactose metabolic imbalance. Strains expressing the intermediate activity alleles of hGALT demonstrated intermediate galactose sensitivity and transient gal-1-P accumulation. Finally, those strains expressing the highest activity alleles of hGALT demonstrated no sensitivity to galactose and no detectable accumulation of gal-1-P. Furthermore, studies of UDP-gal and UDP-glc accumulation in samples prepared from yeast expressing wild-type human GALT versus no GALT showed a specific and significant loss of UDP-gal, but not UDP-glc, only in the GALT-deficient cells in response to the addition of galactose. These results present the first clear evidence in a biochemically and genetically amenable model system of a relationship between hGALT genotype, encoded enzyme activity measured in vitro, aberrant metabolite accumulation, and sensitivity to galactose measuredin vivo. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose toxicity in yeast and perhaps other eukaryotes, including humans. All hGALT mutations were recreated by site-directed mutagenesis of the otherwise wild-type sequence, as described previously (15McClary J.A. Witney F. Geisselsoder J. Biotechniques. 1989; 7: 282-289PubMed Google Scholar). The primers used to generate alleles R67C, L139P, P183T, R201H, R231H, R259W, K285N, E291K, Y323D, and T350A were hGR67CF (5′-GAAGACAGTGCCCTGCCATGACCCTCTC-3′), hGL139PF (5′-GGATGTAACGCCGCCACTCATGTCG-3′), hGP183TF (5′-GCTGTTCTAACACCCACCCCCACT-3′), hGR201HF (5′-GATATTGCCCAGCATGAGGAGCGA-3′), hGR231H (5′-TCAGGAAGGAACATCTGGTCCTAAC-3′), hGR259WF (5′-GCTGCCCCGTTGGCATGTGCGGCGG-3′), hGK285NF (5′-GCTCTTGACCAATTATGACAACCTC-3′), hGE291KF (5′-GACAACCTCTTTAAGACGTCCTTTCC-3′), hGY323DF (5′-CACGCTCATTACGACCCTCCGCTC-3′), hGT350AF (5′-GAGGGACCTCGCCCCTGAGCAGGCT-3′), respectively. All resultant mutant alleles were confirmed by dideoxy sequencing. Recreations of the mutations S135L, V151A, F171S, Q188R, N314D, and R333W have been described previously (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar, 16Crews C. Wilkinson K.D. Wells L. Perkins C. Fridovich-Keil J.L. J. Biol. Chem. 2000; 275: 22847-22853Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 17Elsevier J.P. Wells L. Quimby B.B. Fridovich-Keil J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7166-7171Crossref PubMed Scopus (37) Google Scholar, 18Fridovich-Keil J.L. Quimby B.B. Wells L. Mazur L.A. Elsevier J.P. Biochem. Mol. Med. 1995; 56: 121-130Crossref PubMed Scopus (31) Google Scholar, 19Fridovich-Keil J.L. Langley S.D. Mazur L.A. Lennon J.C. Dembure P.P. Elsas L.J. Am. J. Hum. Genet. 1995; 56: 640-646PubMed Google Scholar). For expression at low copy number, each allele was subcloned using the enzymes EcoRI and SalI into the centromeric yeast vector pMM22 (20Henderson J.M. Wells L. Fridovich-Keil J.L. J. Biol. Chem. 2000; 275: 30088-30091Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). For expression at high copy number, alleles were subcloned into the 2-μm yeast vector pYEP-GAP (a generous gift of Dr. Warren Kruger, Fox Chase Cancer Center). Both plasmids facilitate expression of the introduced open reading frame from the constitutive yeast GAP promoter. All yeast manipulations were carried out according to standard techniques as described previously (21Guthrie C. Fink G. Methods Enzymol. 1991; 194 (; 373–389): 281-301Crossref PubMed Scopus (1102) Google Scholar). All YEP-GAP and MM22 plasmids were transformed into yJFK1, a previously described haploid strain of Saccharomyces cerevisiae deficient in GAL7, the endogenous yeast GALT (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar). Transformants were selected and maintained on the basis of tryptophan prototrophy, conferred by the plasmid. Except where otherwise noted, cells were cultured in media containing dextrose as the sole carbon source to prevent any selective pressure for GALT activity. Soluble cell lysates were prepared from 30-ml cultures grown at 30 °C to A600 = 1.5, essentially as described previously (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar, 22Quimby B.B. Wells L. Wilkinson K.D. Fridovich-Keil J.L. J. Biol. Chem. 1996; 271: 26835-26842Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Briefly, cell pellets were resuspended in 500 μl of lysis buffer (20 mm HEPES, 1 mmdithiothreitol, 0.3 mg/ml bovine serum albumin, 0.3 mmaprotinin, 1 mm pepstatin A, 2 mm antipain, 1 mm phosphoramidon, 0.2 μg/ml chymostatin, 8 mm E64, and 1 mm phenylmethylsulfonyl fluoride) and transferred to 2-ml tubes. 100 μl of acid-washed glass beads (0.5-mm diameter) were added to each tube, and the cells were disrupted with 6 cycles of agitation (45 s on high followed by 45 s on ice) using a multihead Vortex at 4 °C. Each disrupted cell suspension was then transferred to a 1.5-ml tube and centrifuged in a microcentrifuge on high speed for 10 min at 4 °C to pellet insoluble material. Finally, each clarified supernatant was transferred to a fresh tube and assessed for protein concentration using the Bio-Rad protein assay reagent, as recommended by manufacturer, with bovine serum albumin as a standard. GALT activity was determined in soluble yeast lysates as described previously (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar, 22Quimby B.B. Wells L. Wilkinson K.D. Fridovich-Keil J.L. J. Biol. Chem. 1996; 271: 26835-26842Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). For lysates prepared from yeast expressing low copy number plasmids (MM22 derivatives), between 1 and 20 μg of total protein, adjusted to maintain linearity of the assay, was included in each reaction. 1 μg of protein was used for assays of wild-type hGALT under these conditions. For lysates prepared from yeast expressing high copy number plasmids (YEP-GAP derivatives), between 0.3 and 180 μg of total protein, adjusted to maintain linearity of the assay, was included in each reaction. 0.3–0.5 μg of protein was used for assays of wild-type hGALT under these conditions. All assays were performed in triplicate (or greater) as indicated (Table I), representing extracts prepared from independent colonies, and adjusted according to total protein before being normalized to the appropriate wild-type values.Table IActivity assays of soluble lysates from yeast expressing the indicated alleles of human GALTAlleleRelative GALT activity (n)Wild type100.0 ± 5.8 (9)N314D102.5 ± 33.0 (3)E291K62.8 ± 9.8 (3)R201H62.8 ± 9.8 (3)P183T45.2 ± 6.8 (4)T350A9.9 ± 1.3 (3)Y323D9.6 ± 0.9 (3)V151A4.6 ± 1.1 (5)S135L2.7 ± 0.4 (6)R67C2.3 ± 0.4 (3)L139P1.9 ± 0.6 (3)F171S<0.2 ± 0.0 (3)Q188R<0.2 ± 0.0 (3)R231H<0.2 ± 0.0 (6)R259W<0.2 ± 0.0 (12)K285N<0.2 ± 0.0 (5)R333W<0.2 ± 0.0 (9)All values were normalized to the corresponding wild-type GALT activity level and are presented as average ± S.D. (n). Activity levels in extracts demonstrating <5% wild-type activity were determined using cells expressing the indicated hGALT alleles from a high copy number plasmid, whereas those demonstrating ≥∼10% activity were determined using cells expressing the indicated hGALT alleles from centromeric plasmids. In each case activity values associated with the mutant hGALT proteins were normalized against wild-type hGALT expressed from the same vector backbone. Open table in a new tab All values were normalized to the corresponding wild-type GALT activity level and are presented as average ± S.D. (n). Activity levels in extracts demonstrating <5% wild-type activity were determined using cells expressing the indicated hGALT alleles from a high copy number plasmid, whereas those demonstrating ≥∼10% activity were determined using cells expressing the indicated hGALT alleles from centromeric plasmids. In each case activity values associated with the mutant hGALT proteins were normalized against wild-type hGALT expressed from the same vector backbone. Western blot analyses were performed as described previously (18Fridovich-Keil J.L. Quimby B.B. Wells L. Mazur L.A. Elsevier J.P. Biochem. Mol. Med. 1995; 56: 121-130Crossref PubMed Scopus (31) Google Scholar). SDS-polyacrylamide electrophoresis gels to be blotted were loaded with 5 μg/lane of protein representing yeast expressing low copy number plasmids (MM22 derivatives) and either 1 μg (wild-type) or 5 μg (mutants) of protein/lane representing yeast expressing high copy number plasmids (YEP-GAP derivatives). Both wild type and mutant forms of human GALT were detected using a rabbit polyclonal antiserum raised against hexahistidine-tagged hGALT at a dilution of 1:100,000. As a control for loading, blots also were probed with an antiserum against the endogenous yeast protein cyclophilin (at a dilution of 1:30,000) (23Zydowsky L.D. Ho S.I. Baker C.H. McIntyre K. Walsh C.T. Protein Sci. 1992; 1: 961-969Crossref PubMed Scopus (16) Google Scholar). Signals were visualized using a horseradish peroxidase-conjugated antiserum against rabbit Ig (Amersham Pharmacia Biotech, 1:5000 dilution) followed by reaction with the enhanced chemiluminescence (ECL) system from Amersham Pharmacia Biotech, as recommended by the manufacturer. Cultures inoculated from colonies were grown initially in synthetic medium containing 2% dextrose overnight to an A600 between 1 and 2. Cells were then diluted into 6 ml of fresh medium containing 2% glycerol, 2% ethanol in place of dextrose at anA600 = 0.1. These cultures were allowed to grow to an A600 of about 1, at which point they were again diluted in duplicate into 6 ml of fresh synthetic medium containing 2% glycerol, 2% ethanol at an A600= 0.1 to begin growth curves. Time points were taken at 0, 7, 22, and 31 h, at which point the A600 of each culture was close to 0.4. At that point, galactose was added to a final concentration of 0.05% to one culture from each pair, and all tubes were returned to the rotator at 30 °C. Finally, measurements ofA600 were followed for each culture periodically over the next 2–4 days, as indicated (Figs. Figure 2, Figure 3, Figure 4).Figure 2Galactose sensitivity of gal7yeast expressing patient alleles of human GALT. Yeast expressing each of the indicated alleles of hGALT were cultured in synthetic medium containing glycerol/ethanol, with galactose added to 0.05% final concentration at 31 h (arrows). Growth was monitored for each culture by A600. Yeast expressing higher activity alleles of hGALT are presented in theleft-most panel, yeast expressing intermediate activity alleles (+ wild type (WT) as a control) are presented in themiddle panel, and yeast expressing the lowest activity alleles (+WT as a control) are presented in theright-most panel. All values plotted represent averages ± S.D. (n = 3). Parallel samples of all cultures maintained in medium containing dextrose grew well (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Impact of galactose exposure on accumulation of UDP-gal and UDP-glc in yeast. As in Fig. 3, yeast expressing either no GALT or wild-type hGALT were cultured in synthetic medium containing glycerol/ethanol, with galactose added to half of the cultures at 0.05% final concentration at 40 h (arrows). In addition to monitoringA600 at the indicated times (panels Aand B), samples of each culture also were harvested and analyzed for intracellular levels of gal-1-P (panels C andD), UDP-gal (panels E and F), and UDP-glc (panels G and H). In allpanels, samples representing cultures with galactose are represented by open circles, and samples representing cultures without galactose are represented by filled circles. All values plotted represent average ± S.D. (n = 3). In panel C, samples representing yeast cultured in both the presence and absence of galactose are plotted, although only one set is visible because the values at each time point were coincident (all zero). Similarly, gal-1-P measurements at the 40-h time point (panel D) representing yeast cultured in both the presence and absence of galactose were both zero, so only one is visible in the figure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Samples were prepared from duplicate 30-ml cultures of cells grown as described above (before the addition of galactose). At 31 h after inoculation, a 6-ml sample was removed from each culture (zero time point), and galactose was added to the remaining volume of one culture from each pair to a final concentration of 0.05%. At 7, 15, and 63 h after the addition of galactose, 6-ml samples from each culture were harvested, pelleted, and frozen. Finally, cell pellets were lysed as described above, except that protease inhibitors were not included in the lysis buffer. Protein concentrations were measured, as described above, using the Bio-Rad protein assay reagent as recommended by manufacturer, with bovine serum albumin as a standard. Next, each lysate was cleared of proteins by vigorous extraction with methanol (500 μl of methanol/200 μl of aqueous sample) followed by centrifugation to pellet the protein precipitate. Finally, each clarified supernatant was transferred to a fresh 1.5-ml tube and dried under vacuum. Pellets were resuspended in sterile, deionized water for further analysis. Gal-1-P levels were quantitated using a coupled spectrophotometric assay described previously (17Elsevier J.P. Wells L. Quimby B.B. Fridovich-Keil J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7166-7171Crossref PubMed Scopus (37) Google Scholar, 22Quimby B.B. Wells L. Wilkinson K.D. Fridovich-Keil J.L. J. Biol. Chem. 1996; 271: 26835-26842Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) with slight modifications. In particular, the assay buffer contained 100 mmglycylglycine, pH 8.7, 6 mm dithiothreitol, 5 μm glucose-1,6-diphosphate, 5 mmMgCl2, 0.8 mm NADP, 0.1 μg of phosphoglucomutase, and 0.06 μg of glucose-6-phosphate dehydrogenase in a total reaction volume of 400 μl. All assays were carried out using 300 ng of purified His6-tagged hGALT isolated from a yeast expression system. A standard curve was generated using UDP-glucose at a concentration of 0.6 mm with varying concentrations of galactose 1-phosphate (0.075–1.2 mm). To determine the levels of galactose 1-phosphate present in each test sample, 50 μl of deproteinated extract (representing 20–416 μg of original lysate protein) were used in place of a known quantity of galactose 1-phosphate so that the amount of gal-1-P present in that sample could be determined by interpolation from the standard curve. Final values presented (Figs. 3 and 4) were normalized according to total protein in each original extract. To ensure that our measurements of gal-1-P were comparable with those of other groups using the method of Bergmeyer (24Bergmeyer, H. (ed) (1974)Methods of Enzymatic Analysis, pp. 1291-1295,Academic Press, Inc., New YorkGoogle Scholar), which utilizes alkaline phosphatase and galactose dehydrogenase, we assayed a set of test samples, some with high gal-1-P and others with low gal-1-P, by both methods. In all cases comparable values were obtained from both assays (data not shown). Samples were prepared from cultures of yeast expressing either no GALT or wild-type human GALT grown in the presence versus absence of galactose, harvested, lysed, and cleared of proteins, as described above. UDP-gal was measured against a standard curve established using a coupled reaction with purified UDP-gal-4 epimerase (a kind gift of Drs. Jim Thoden and Hazel Holden, University of Wisconsin, Madison, WI) and UDP-glc dehydrogenase (Sigma), as described previously (25Wohlers T.M. Fridovich-Keil J.L. J. Inherited Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (46) Google Scholar). UDP-glc was measured directly in each sample in the absence of epimerase using UDP-glc dehydrogenase (Sigma) and also quantitated by comparison with a standard curve. Final values presented (Fig. 4) were normalized according to total protein in each original extract. We have used site-directed mutagenesis of the wild-type human GALT sequence to recreate each of 16 naturally occurring mutations: R67C, S135L, L139P, V151A, F171S, P183T, Q188R, R201H, R231H, R259W, K285N, E291K, N314D, Y323D, R333W, T350A. After confirmation, each allele was introduced into both low copy number (CEN, MM22) and high copy number (2 μm, YEP-GAP) yeast expression plasmids containing the constitutive GAP promoter and transformed into the previously described null background strain ofS. cerevisiae, yJFK1 (14Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar). Plasmids encoding the wild-type hGALT sequence and empty plasmids alone were also included in all experiments as positive and negative controls, respectively. Unless otherwise noted, all cultures were maintained in dextrose-containing medium to prevent selective pressure based on encoded hGALT activity. To confirm expression and to determine the relative abundance of each substituted hGALT protein in yeast, soluble lysates prepared from cells expressing each hGALT allele from a centromeric plasmid were subjected to Western blot analysis with the rabbit polyclonal anti-hGALT antiserum, EU70. As a control for loading of lanes, each filter was also probed with a polyclonal antiserum that recognizes yeast cyclophilin (23Zydowsky L.D. Ho S.I. Baker C.H. McIntyre K. Walsh C.T. Protein Sci. 199
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