The
FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of
flavin adenine dinucleotide (
FAD) and an increased level of
flavin mononucleotide (
FMN) in mitochondria. The restoration of respiratory capability by
FAD1 is shown to be due to extragenic suppression.
FAD1 codes for an essential yeast
protein, since disruption of the gene induces a lethal phenotype. The
FAD1 product has been inferred to be yeast
FAD synthetase, an
enzyme that adenylates
FMN to
FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with
FAD1 on a multicopy plasmid displays an increase in
FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the
FAD1 product exhibits low but significant primary sequence similarity to
sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of
FAD synthetase. The lower mitochondrial concentration of
FAD in G178 mutants is proposed to be caused by an inefficient exchange of external
FAD for internal
FMN. This is supported by the absence of
FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial
riboflavin kinase, the preceding
enzyme in the biosynthetic pathway. A lesion in mitochondrial import of
FAD would account for the higher concentration of mitochondrial
FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of
FAD1 to suppress impaired transport of
FAD is explained by mislocalization of the
synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial
FAD synthetase activity in S. cerevisiae transformed with
FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.