Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.
The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.
Hosaka
K
K
Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Murakami
T
T
Kodaki
T
T
Nikawa
J
J
Yamashita
S
S
D000906:Antibodies; D015152:Blotting, Northern; D002794:Choline / Q000494:pharmacology*; D002797:Choline Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004794:Enzyme Repression; D005800:Genes, Fungal; D007118:Immunoassay; D007294:Inositol / Q000494:pharmacology*; D010770:Phosphotransferases / Q000096:biosynthesis*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D000906:Antibodies; D007294:Inositol; D010770:Phosphotransferases; D002797:Choline Kinase; D002794:Choline