Regulation of hypoxanthine transport in Neurospora crassa

Abstract

Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.

Developmental-stage-dependent adenine transport in Neurospora crassa

“…4. As seen in the figure, the ad-8 strain could not accumulate hypoxanthine at all from the medium, which is in agreement with earlier reports (6,15), whereas the ad-4 strain had lowered ability, compared with the wild type. In general, even in the wild type the rate of hypoxanthine accumulation through the general transport system was lower than that of adenine accumulation, which may be a reflection of the differences in the affinity of this system for the two purine bases.…”

“…6). Considering previous reports that growth in ammoniumcontaining medium stimulated hypoxanthine transport (i.e., the general purine system) in Neurospora (15,16), the present finding may suggest that ammonium is also required for the expression of the adenine-specific system. The possibility, however, that the formation of the second system is linked to the germination process cannot be ruled out.…”

“…The observed differences in Vinax and similarities in K,,, between the wild type and ad-8 (for the low concentration range) suggest that the ad-8 strain has low levels of the general purine permease or that the activity is noncompetitively inhibited. It is possible that the compound that this mutant accumulates or excretes into the medium, namely, inosine or hypoxanthine (13,15), may be responsible for the inhibition of adenine transport in the lower concentration range. In such a situation, however, if hypoxanthine is the inhibitory compound, the double reciprocal plot of adenine transport at low concentrations should resemble a competitive inhibition plot, which is not evident.…”

Nature of 6-methylpurine inhibition and characterization of two 6-methylpurine-resistant mutants of Neurospora crassa

“…It is posible that the reduction of HPRTase activity in these strains is simlar to that observed in ad-8 mutants of Neurospora. The primary leon of the ad-8 mutant is the loss of activity of adenylosuccinate synthetase (22), but the mutants also posess a defective general purine permease and HPRTase (29). It seems that in the present case the reduction in the level of HPRTase activity in the 6-methylpurine-resistant mutants is consequent to the defect that led to the 6methylpurine resistance rather than the cause of it.…”

“…In our experience, even short-term pulse-labeling studies of purine bases always yielded purine nucleotides in subsequent extractions of the cells (24,25). Sabina et al (29) found that an adenine-requiring mutant ofNeurospora (ad-8) not only could not transport hypoxanthine, but its hypoxanthine PRTase (EC 2.4.2.8; HPRTase) activity was very low. The last two observations could mean either that a group tranalocation process is operative in Neurospora for purine bases or that purine bases are metabolized to nucleotide form very rapidly upon entry into the cells and that both transport and phosphoribosylation may be regulated by the same intracellular accumulation products.…”