Isolation and characterization of recessive, constitutive mutations for repressible acid phosphatase synthesis in Saccharomyces cerevisiae.

Two new classes of mutants containing recessive constitutive mutations, phoT and phoU, that affect the repressible acid phosphatase (EC 3.1.3.2) in Saccharomyces cerevisiae were isolated along with many previously known phoR mutants. These loci segregated independently from each other, from the phoS gene, and from another regulatory gene, phoD, that exerts positive control for acid phosphatase synthesis. The phoR and phoU mutations showed the same genetic behavior in the double mutants, which also contained the phoS or phoD mutation. In contrast, the phoT mutation could not suppress the phoS mutation, which caused a loss of enzyme activity. Many mutant alleles of phoR and phoU were found to be temperature sensitive (ts), whereas those of phoT were not. These ts mutants were constitutive at 35 C but severely repressible at 25 C. These facts strongly suggest that both the phoR and phoU genes are cooperatively concerned with the production of the repressor, whereas the phoT gene might be involved in another mechanism distinct from that in which phoR and phoU are involved. No single mutation of phoR, phoT, or phoU result in an enzyme level comparable to that of fully derepressed enzyme activities, and the temperature sensitivity of the ts phoR and ts phoU mutations in such combinations almost disappeared. In addition to these observations, since the ts phoR phoS and ts phoU phoS double mutants showed some enzyme synthesis at 25 C under derepressing conditions, a defect in the ts mutant repressors was strongly suggested, even at 25 C.

Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strain H-42 seems to have two kinds of acid phosphatase: one which is constitutive and one which is repressible by inorganic phosphate. The constitutive enzyme was significantly unstable to heat inactivation, and its K(m) of 9.1 x 10(-4)m for p-nitrophenylphosphate was higher than that of the repressible enzyme (2.4 x 10(-4)m). The constitutive and the repressible acid phosphatases are specified by the phoC gene and by the phoB, phoD, or phoE gene, respectively. Results of tetrad analysis suggested that the phoC and phoE genes are linked to the lys2 locus on chromosome II. Since both repressible acid and alkaline phosphatases were affected simultaneously in the phoR, phoD, and phoS mutants, it was concluded that these enzymes were under the same regulatory mechanism or that they shared a common polypeptide. The phoR mutant produced acid phosphatase constitutively, and the phoR mutant allele was recessive to its wild-type counterpart. The phoS mutant showed a phenotype similar to that of a mutant defective in one of the phoB, phoD, or phoE genes. However, the results of genetic analysis of the phoS mutant clearly indicated that the phoS gene is not a structural gene for either of the repressible acid and alkaline phosphatases, but is a kind of regulatory gene. According to the proposed model, the phoS gene controls the expression of the phoR gene, and inorganic phosphate would act primarily as an inducer for the formation of the phoR product which represses phosphatase synthesis.