Ureidosuccinate is transported by the allantoate transport system in Saccharomyces cerevisiae.

The regulatory characteristics exhibited by ureidosuccinate transport in Saccharomyces cerevisiae led us to hypothesize that this biosynthetic intermediate was transported via the degradative allantoate transport system. The hypothesis was verified by the finding that neither dal5 nor urep1 mutant strains could transport allantoate or ureidosuccinate. Mutations in the two loci were tightly linked and failed to complement one another, suggesting that they were allelic. The use of a common transport system for accumulation of both biosynthetic and degradative metabolites explains the paradoxical characteristics observed for control of ureidosuccinate and allantoate transport.

Location of the genes that control induction of the allantoin-degrading enzymes in Saccharomyces cerevisiae.

In an effort to understand the regulation of allantoin degradation in Saccharomyces cerevisiae, we isolated two classes of mutants, each defective in the induction process associated with production of the pathway enzymes. Mutation at one locus (DAL80) results in constitutive expression of the genes involved in allantoin catabolism. Mutation at the second locus (DAL-81) results in the loss of ability to induce these enzymes. This report describes genetic data indicating that the DAL80 and DAL81 loci are situated approximately 13 cM from the centromere on the right arm of chromosome XI and 9 cM proximal to the DAL1 locus on chromosome IX, respectively.

Pleiotropic control of five eucaryotic genes by multiple regulatory elements.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.

Structural analysis of the dur loci in S. cerevisiae: two domains of a single multifunctional gene.

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.

Allantoate transport in Saccharomyces cerevisiae.

Allantoate uptake appears to be mediated by an energy-dependent active transport system with an apparent Michaelis constant of about 50 microM. Cells were able to accumulate allantoate to greater than 3,000 times the extracellular concentration. The rate of accumulation was maximum at pH 5.7 to 5.8. The energy source for allantoate uptake is probably different from that for uptake of the other allantoin pathway intermediates. The latter systems are inhibited by arsenate, fluoride, dinitrophenol, and carboxyl cyanide-m-chlorophenyl hydrazone, whereas allantoate accumulation was sensitive to only dinitrophenol and carboxyl cyanide-m-chlorophenyl hydrazone. Efflux of preloaded allanotate did not occur at detectable levels. However, exchange of intra- and extracellular allantoate was found to occur very slowly. The latter two characteristics are shared with the allantoin uptake system and may result from the sequestering of intracellular allantoate within the cell vacuole. During the course of these studies, we found that, contrary to earlier reports, the reaction catalyzed by allantoinase is freely reversible.

A cluster of three genes responsible for allantoin degradation in Saccharomyces cerevisiae.

Mutant strains of Saccharomyces cerevisiae unable to utilize allantoin as sole nitrogen source were isolated and divided into three groups on the basis of their biochemical and genetic characteristics. The three loci associated with these mutant classes were designated dal1 (allantoinase minus), dal2 (allantoicase minus) and dal4 (allantoin transport minus). All three loci are located in a cluster that is proximal to the lys1 locus on the right arm of chromosome IX. The gene order and intergenic distances were estimated to be: dal1--2.5 cM--dal4--1.9cM--dal2--4.6cM-lys1.

Induction and inhibition of the allantoin permease in Saccharomyces cerevisiae.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.