Expression of UDP-glucuronosyltransferase cDNA in Saccharomyces cerevisiae as a membrane-bound and as a cytosolic form.
The mouse clone UDPGTm-1 encodes a UDP-glucuronosyltransferase enzyme which was isolated from a lambda gt11 cDNA library constructed with phenobarbital-induced liver mRNA [Kimura, T., & Owens, I. S. (1987) Eur. J. Biochem. 168, 515-521]. In order to establish substrate specificity, UDPGTm-1 was inserted into the yeast vector pEVP11 and expressed in Saccharomyces cerevisiae strain AH22. Cells transformed with the expression unit pUDPGTm-1c (insert in correct orientation with respect to promoter) stably transcribe the transferase cDNA. Consistent with the presence of mRNA, pUDPGTm-1c-transformed AH22 cells synthesize a transferase protein with Mr congruent to 51,000 by Western immunoblot analysis. The membrane-bound transferase expressed in yeast in glycosylated as indicated by its enhanced electrophoretic mobility in a SDS-polyacrylamide gel following endoglycosidase H treatment and detection by Western immunoblot analysis. A survey, using 12 aglycons in an assay with microsomes from cells which express the protein, shows preferential glucuronidation of naphthol and estrone followed by p-nitrophenol. Testosterone, phenolphthalein, dihydrotestosterone, androsterone, and 4-methylumbelliferone are conjugated at an intermediate level. There is barely detectable glucuronidation of 3-hydroxy- and 9-hydroxybenzo[a]pyrene and no detectable conversion of morphine or lithocholic acid. The truncated cDNA (lacking the putative membrane-insertion signal-peptide coding sequence, but with a newly adapted translation-start codon) is ligated into pAAH5 and is expressed as a cytosolic transferase form in the protease-deficient ZA521 strain of S. cerevisiae. The Mr congruent to 51,000-52,000 is similar to that seen in microsomes from AH22 cells where the protein is presumably processed as it is inserted into the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Toghrol
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Section on Drug Biotransformation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Kimura
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Owens
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D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology*; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004247:DNA; D014453:Glucuronosyltransferase