Expression of UDP-glucuronosyltransferase cDNA in Saccharomyces cerevisiae as a membrane-bound and as a cytosolic form

Biochemistry. 1990 Mar 6;29(9):2349-56. doi: 10.1021/bi00461a020.

Abstract

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)

MeSH terms

  • Base Sequence
  • Blotting, Western
  • Cell Membrane / enzymology*
  • Cytosol / enzymology*
  • DNA / genetics
  • Gene Expression*
  • Genes, Fungal
  • Glucuronosyltransferase / biosynthesis
  • Glucuronosyltransferase / genetics*
  • Glucuronosyltransferase / metabolism
  • Glycosylation
  • Molecular Sequence Data
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Substrate Specificity
  • Transformation, Genetic

Substances

  • DNA
  • Glucuronosyltransferase