Glucose-induced phosphorylation of the MDH2 isozyme of malate dehydrogenase in Saccharomyces cerevisiae.
The cytosolic isozyme of malate dehydrogenase, MDH2, was previously shown to be subject to rapid inactivation and proteolysis following the addition of glucose to yeast cultures growing on nonfermentable carbon sources. In this report, we show that MDH2 is phosphorylated during the process of glucose-induced degradation. A truncated active form of MDH2 lacking the first 12 residues of the amino terminus was previously found to be resistant to glucose-induced degradation and, as shown in this study, is not subject to phosphorylation. Site-directed mutagenesis was conducted to change Ser-12 in the authentic enzyme to Ala-12 and to Asp-12. The S12A substitution has little effect on glucose-induced phosphorylation and degradation, whereas the enzyme with the S12D substitution is subject to phosphorylation and inactivation but not to rapid degradation. This provides clear evidence that inactivation is not simply a result of degradation. Additional mutagenesis was conducted to change His-214, a critical active site residue, to Leu-214. Analysis of expression of full-length and truncated forms of the H214L enzyme demonstrated that catalytic inactivity is not a prerequisite for degradation and confirmed an essential role for the amino terminus of the authentic enzyme in this phenomenon.
Minard
K I
KI
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
McAlister-Henn
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L
D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000737:chemistry; D005947:Glucose / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D017931:DNA Primers; D007527:Isoenzymes; D010768:Phosphoserine; D008291:Malate Dehydrogenase; D005947:Glucose