Mutations in Neurospora crassa which affect multiple amino acid transport systems

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize l-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3.Transport of l-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

Characterization of 2-aminoisobutyric acid transport in Neurospora crassa: a general amino acid permease-specific substrate.

We report the characterization of an amino acid 2-aminoisobutyric acid was transported solely by the general amino acid permease and not by the neutral amino acid permease. Furthermore, this substrate was not metabolized after transport. The potential for a system-specific nonmetabolizable substrate as a tool in the analysis of amino acid transport and its regulation is discussed.     

Fructose transport in Neurospora crassa.

A specific fructose uptake system (Km = 0.4 mM) appeared in Neurospora crassa when glucose-grown mycelia were starved. Fructose uptake had kinetics different from those of intramycelial fructose phosphorylation, and uptake appeared to be carrier mediated. The only sugar which competitively inhibited fructose uptake was L-sorbose (Ki = 9 mM). Glucose, 2-deoxyglucose, mannose, and 3-O-methyl glucose were noncompetitive inhibitors of fructose uptake. Incubation of glucose-grown mycelia with glucose, 2-deoxyglucose, or mannose prevented derepression of the fructose transport system, whereas incubation with 3-O-methyl glucose caused the appearance of five times as much fructose uptake activity as did starvation conditions.     

Physiological and regulatory properties of the general amino acid transport system of Neurospora crassa.

The fundamental properties of the general amino acid transport system of Neurospora crassa were investigated in the conidial stage of the life cycle. The transport activity was found to be under genetic control, and an isogenic set of mutants deficient for the neutral, basic, or general amino acid transport systems and combinations thereof was constructed and used for analyzing the properties specific to the general permease. Amino acid transport by this system was found to be a carrier-mediated active process with broad specificity for the neutral and basic amino acids. Kinetic analysis revealed that a common binding site functioned to transport both neutral and basic amino acids and that the permease had a high affinity for its substrates. The kinetic parameters Km, Vmax, and Ki were defined for several substrates. Two modes of regulation were detected: substrate inhibition and ammonium repression. Activity of the general system was enhanced by the removal of ammonium ions from the incubation medium with a concomitant decline in either neutral or basic permease activity, suggesting that a common component exists between the neutral and the general systems and between the basic and the general systems.     

Characterization of the glucose transport systems in Neurospora crassa sl.

Neurospora crassa sl, a mutant that lacks a rigid cell wall, exhibits transport systems for glucose similar to those of wild-type strain 1A. When the orgnism is grown in a medium containing 50 mM glucose as the carbon source, glucose is transported primarily by a glucose-facilitated diffusion system (GluI). When it is grown in a medium with little or no glucose present, a glucose active transport system (Glu II) is expressed. Both of these systems are similar kinetically to those in the wild type. Significant differences do exist between strains sl and 1A with respect to genetic regulation of the glucose active transport system.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Genetic control of amino acid permeability in Neurospora crassa

Lester, Gabriel (Reed College, Portland, Ore.). Genetic control of amino acid permeability in Neurospora crassa. J. Bacteriol. 91:677-684. 1966.-Strains of Neurospora crassa resistant to 4-methyltryptophan (4-MT) were isolated from populations of conidia exposed to ultraviolet light. In genetic crosses, 4-MT resistance behaved as a single-gene difference. Resistance to 4-MT could not be attributed to a relaxation of control of the formation or the activity of the enzymes of tryptophan biosynthesis. Growth studies involving tryptophan auxotrophs carrying the aberrant mt gene and uptake studies with normal and 4-MT-resistant strains showed that 4-MT resistance could be attributed to an inability of 4-MT-resistant strains to take up tryptophan and its methyl analogues. The mt gene is not specific for tryptophan; strains resistant to 4-MT are also resistant to ethionine, and they have a markedly reduced ability to take up serine, leucine, and alpha-aminoisobutyric acid. No difference was observed between strains carrying either mt allele in their ability to take up glucose; also, the uptake of anthranilic acid or of indole was not sufficiently impaired by the aberrant mt gene to prevent these tryptophan precursors from satisfying the nutritional requirement of certain tryptophan auxotrophs. The role of the mt gene in determining the permeability of N. crassa to amino acids is discussed.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.