Abstract
aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.
Highlights
Saccharomyces cerevisiae pet mutants have been used to study a broad range of problems related to mitochondrial function and biogenesis (1)
The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type
We proposed that in addition to its known function in riboflavin synthesis, RIB3 functions in expression of mitochondrial respiration
Summary
Saccharomyces cerevisiae pet mutants have been used to study a broad range of problems related to mitochondrial function and biogenesis (1) Such mutants have been assigned to the following phenotypic classes based on their compositions of respiratory chain components: 1) mutants defective in individual carriers of the respiratory chain, 2) mutants having a normal set of respiratory enzymes (e.g. cytochrome spectra and ATPase), and 3) mutants with partial or severe pleiotropic deficiencies in respiratory chain components. Functional analysis of the latter is difficult because the phenotype fails to provide clues as to the nature of the biochemical lesion. We present evidence that the phenotype of aE280/U1 is not related to a flavin nucleotide deficiency and propose that in addition to its involvement in riboflavin synthesis, DHBP synthase has another function essential for expression of respiration in yeast
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