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

Abstract

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...

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“…(crssa. These are the basic amino acid permease (183,197,221) and the general amino acid permease (68,179,182,195), deficient in strains carrying the bhit and ptn,g' mrUtations,…”

Continuous imaging of amino‐acid translocation in intact mycelia of Phanerochaete velutina reveals rapid, pulsatile fluxes

“…This approach only provides unequivocal evidence for N-translocation if the compound selected reliably follows the normal pathway for natural Ncompounds and is not metabolized by the fungus. One such compound is 2-amino[1-14 C]isobutyric acid (AIB), a methylated analogue of alanine, which is actively transported into the cell by amino-acid transport proteins (Ogilvie-Villa et al , 1981) and accumulated without being metabolized or incorporated into protein (Kim & Roon, 1982). It is translocated by many fungal species without being metabolized ( Watkinson, 1984a;Lilly et al , 1990;Olsson & Gray, 1998).…”

Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa

“…In the filamentous fungus Neurospora crassa, transport is accomplished by three constitutive amino acid permeases and three specialized permeases. The constitutive permeases include a neutral amino acid-specific (N) system, a basic amino acid-specific (B) system, and a general (G) system that can transport all classes of amino acid (5,7,18,20,22,23,37). Three additional systems that function during advanced stages of development have been proposed for the transport of the imino acid proline, of acidic amino acids, and of the sulfurcontaining amino acid methionine during sulfur limitation (10,24,25,38,40).…”