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 Ki, 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.

A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed.

The growth of the pm nbg mutant strain of Neurospora crassa was inhibited by the amino acid analog para-fluorophenylalanine despite the fact that none of the three constitutive amino acid permeases is functional in this strain. This observation led to the detection of both a deaminase which was released into the growth medium in response topara-fluorophenylalanine and a keto acid transport system which allowed entry of the resulting keto acid into the cell. The transported keto acid was recovered in cellular protein, suggesting its regeneration as the amino acid. The cooperative activity of these two systems represents an additional mechanism for the intracellular accumulation of amino acids, which is distinct from the known amino acid permeases. Amino acid transport in the ascomycete Neurospora crassa is accomplished by means of the following three constitutive amino acid permeases: (i) the neutral pennease system, which transports the class of amino acids with neutral

We report the characterization of an amino acid permease-specific substrate for Neurospora crassa. The neutral amino acid 2-aminoisobutyric acid was transported solely by the general amino acid permease and not by the neutral amino acid permease. Furthernore, 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.Several laboratories have been involved with the analysis of amino acid transport in the fungus Neurospora crassa. The early goals of this research were to determine the number and types of systems which were responsible for transporting amino acids. This approach defined the strategy for amino acid transport by N. crassa as involving a limited number of constitutive permeases which transport families of structurally related amino acids: a neutral (N) system which is specific for neutral aliphatic or aromatic amino acids, a basic (B) system which transports positively charged amino acids, and a general (G) system which handles all classes of amino acids (1, 2, 4-7, 9-11, 14).Each family of amino acids is, therefore, transported both by the family-specific system and by the G system. Such overlap in substrate specificity between systems has made it difficult to ascribe particular properties to single systems. One approach to this problem involves the construction of mutant strains defective for the activity of one or more transport systems so that only the system of interest remains active. This approach has been very useful in defining the number of transport systems that exist and the physiological and kinetic properties of each. A second approach involves the identification and use of a system-specific amino acid substrate to monitor the activity of the permease of interest without eliminating the activities of the other perneases. This approach allows one to detect interactions among functional systems, an advantage not possible with the mutant approach. In addition, a permease-specific substrate is a useful tool, in combination with the mutant approach, for molecular studies of the components which comprise a particular transport system.We describe here the transport of the amino acid 2-aminoisobutyric acid (AIB) and its properties as a general amino acid permease-specific substrate. MATERLS AND METHODSStrains and growth of cultures. The wild-type strain 74a (FGSC 988) was obtained from the Fungal The pmn, pmb, and pmg strains are defective for the neutral amino acid-specific permease, the basic amino acid-specific permease, and the general amino acid permease, respectively, and are isogenic with the wild type. Each of these strains is available from the authors. All strains were maintained in lx Vogel medium N supplemented with 2% sucrose and solidified with 1.5% agar as previously described (2,13).Amino acid transport assays. For the conidial assays, cells were incubated at 25°C (0.1 mg of dry weight per ml of incubation medium, final concentration) in lx Vog...

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is pnder 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.