Abstract
The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.
Highlights
The compound 5-phospho-D-ribosyl ␣-1-diphosphate (PRPP)1 is an important component of the metabolism of most organisms
Complementation was analyzed in the following strains: HO773, which contains a large deletion of the prsEc gene, resulting in lack of PRPP synthase activity; HO2264, HO2268, HO2480, HO2284, and HO2481, which had the prsEc gene replaced by the S. cerevisiae PRS1, PRS5, PRS2, PRS3, and PRS4 gene, respectively
I used an E. coli ⌬prs strain with no endogenous PRPP synthase activity to detect the formation of active PRPP synthase specified by the five PRS genes
Summary
The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1–5), which specify PRPP synthase subunits 1–5. Expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. The data indicate that certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. Except for certain specialized mutants of Escherichia coli, all freeliving organisms contain at least one gene encoding PRPP synthase. The yeast Saccharomyces cerevisiae contains five PRPP synthase-homologous genes, PRS1–5, encoding PRPP synthase subunits 1–5, respectively. S. cerevisiae PRPP synthase subunits 1 and 5 contain additional amino acids, which constitute non-homologous regions (NHRs). The S. cerevisiae PRS genes may be designated as PRSSc and that of E. coli may be designated as prsEc
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