Expression and function of heterologous forms of malate dehydrogenase in yeast.
The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.
Steffan
J S
JS
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
Minard
K I
KI
McAlister-Henn
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005816:Genetic Complementation Test; D066298:In Vitro Techniques; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010101:Oxygen Consumption; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity
D008291:Malate Dehydrogenase