Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants.
A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.
Beckmann
J D
JD
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Ljungdahl
P O
PO
Trumpower
B L
BL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D014450:Electron Transport Complex III; D005816:Genetic Complementation Test; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008667:Metalloproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III