A. Gib DeBusk

Molecular transport in Neurospora crassa I. Biochemical properties of a phenylalanine permease

Abstract 1. 1. Neurospora crassa has a constitutive transport system for phenylalanine which is energy dependent, temperature dependent, shows stereospecificity, has an optimum pH of 5.0–5.5 and a Km of 10−4 M. 2. 2. The system can transport phenylalanine against a concentration gradient and produce a 700-fold concentration over the exterior concentration. This concentration gradient can be maintained for long periods of time and is energy-requiring. 3. 3. A decrease in the rate of transport and diminution of the free amino acid pool in the presence of glucose has been observed. 4. 4. The action of the permease system can be competitively inhibited by several amino acids. 5. 5. The free amino acid pool has been shown to be large and expandable but has a definite size limit. 6. 6. Competition between phenylalanine and other amino acids for both the permease and occupation of the free amino pool are discussed.

Molecular transport: I. In vivo studies of transport mutants of Neurospora crassa with altered amino acid competition patterns

Abstract The active transport of metabolites by Neurospora crassa conidia has been studied by both kinetic and genetic methods. Transport, reported to be dependent on temperature, pH, and an energy source, is shown to accumulate amino acids by (1) a system specific for neutral-aromatic amino acids, the neutral transport system; (2) a system specific for basic amino acids, the basic transport system; and (3) a non-specific general amino acid transport system. Transport by any of these systems, separately or in combination, results in the accumulation of the respective amino acid against a concentration gradient. The p -fluorophenylalanine-resistant mutant, Pm − N 22 , is deficient in transport of l -phenylalanine, but not l -arginine. Analogous to this is the canavanine-resistant mutant, Pm − B 37 , which is deficient in transport of l -arginine, but not l -phenylalanine. The double mutant, Pm − NB (derived from a cross of Pm − N 22 and Pm − B 37 ) has reduced transport of both l -arginine and l -phenylalanine. This double mutant retains some residual transport activity for both the basic and neutral amino acids. The kinetic properties of this residual activity indicate a capability for transport of either neutral or basic amino acids and further suggests that it is under separate genetic control.

Molecular transport. I. In-vitro studies of isolated glycoprotein subunits of the amino acid transport system of Neurospora crassa conidia.

Abstract Neurospora crassa conidia have constitutive transport systems for neutral and basic amino acids. KCl-extracted conidia show reduced l -arginine transport. KCl extracts contain several molecular species, some of which will bind l -amino acids. Purification of these extracts by affinity chromatography yields two glycoprotein species with a relatively high affinity for l -arginine. The chromatographic disappearance of one glycoprotein species is associated with a single gene mutation resulting in reduction of l -arginine transport in vivo . Disappearance of the second glycoprotein species from the chromatography profile is associated with modification of either of two, unlinked transport loci. Initial biochemical characterization of both molecular species indicate a weight composition of approximately 50% carbohydrate and 50% protein. Disc electrophoresis of the glycoprotein species associated with the single gene transport mutation yields a single band of both amido black and periodic acid-Schiff reagent staining material.Abstract Neurospora crassa conidia have constitutive transport systems for neutral and basic amino acids. KCl-extracted conidia show reduced l -arginine transport. KCl extracts contain several molecular species, some of which will bind l -amino acids. Purification of these extracts by affinity chromatography yields two glycoprotein species with a relatively high affinity for l -arginine. The chromatographic disappearance of one glycoprotein species is associated with a single gene mutation resulting in reduction of l -arginine transport in vivo . Disappearance of the second glycoprotein species from the chromatography profile is associated with modification of either of two, unlinked transport loci. Initial biochemical characterization of both molecular species indicate a weight composition of approximately 50% carbohydrate and 50% protein. Disc electrophoresis of the glycoprotein species associated with the single gene transport mutation yields a single band of both amido black and periodic acid-Schiff reagent staining material.

The β-galactosidase system of Neurospora crassa: I. Purification and properties of the pH 4.2 enzyme

Abstract Extracts of Neurospora crassa contain a β-galactosidase with a pH optimum of 7.5 and a β-galactosidase with a pH optimum of 4.2. The pH 4.2 enzyme was purified by ammonium sulfate precipitation and cation-exchange chromatography. The physical properties of the purified enzyme were similar to those of the nonpurified enzyme as reported previously, but arginine was absent from the former.

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogels medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.

The kinetics of L-aspartate transport in Neurospora crassa conidia.

Transport of the acidic amino acids by Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The neutral transport system is shown to possess “high affinity”, but low transport capacity for the acidic amino acids. The general transport system is shown to have “low affinity” and high transport capacity for these amino acids. The non-linearity of a Lineweaver-Burk plot of aspartate transport by the wild type has been shown to result from an overlapping of two transport systems for accumulation of this amino acid. Removal, by mutation, of the neutral transport system restores the transport kinetics to a linear function. The combined, and separate, transport activities are shown to be stereospecific, preferring the l-stereoisomeric form over the d-form, and to transport the neutrally charged species of l-aspartate and l-glutamate. The transport activity is dependent on pH, and the V, but not the C12, constant of the general transport system is pH dependent. Two models for amino acid transport are presented as possible mechanisms to explain the observed kinetics. One model involves three genetically distinct transport systems. The second model utilizes a single “allosteric permease” to account for the kinetic data.

Active transport of l-aspartic acid in Neurospora crassa

Abstract Transport of l -aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l -aspartic acid against a gradient. Mutants deficient in transport of l -aspartic acid are described.

The β-galactosidase system of Neurospora crassa: II. Extracellular nature of the pH 4.2 enzyme

Abstract β-Galactosidase from Neurospora crassa occurs in two forms with pH optima at 7.5 and 4.2. When grown on lactose, wild type strains release the pH 4.2 enzyme to the media. The extracellular enzyme exists in two electrophoretic forms one of which has a pH optimum of 4.5. The pH 4.5 enzyme is the major extracellular form in the early stages of induction and is released from mycelia by mild extraction. The pH 7.5 enzyme is strongly bound to the cell as is the pH 4.2 enzyme prior to its release to the medium. The pH 4.5 enzyme appears to be converted to the pH 4.2 enzyme by dialysis.

Linear and ‘lasso-like’ structures of mitochondrial DNA from Pennisetum typhoides

Previously unidentified structures of plant mitochondrial DNA, namely intact linear molecules and ‘lasso‐like’ structures, are described. The genomic‐size circular DNA is concluded to be an end product of the progression of the ‘lasso’ structure. These findings give insight into the heterogeneity of mitochondrial DNA unique to higher plants.

Transport of arginine by an in vitro system

Abstract Cell surface glycoproteins of Neurospora crassa conidia have been shown to bind amino acids and to be genetically associated with the previously defined amino acid transport systems of that organism. L-arginine does not readily permeate a film of Neurospora conidial lipids. Addition of glycoprotein extracts from Neurospora to the lipid film enhances permeation of arginine at an initial rate 1000 times the rate of permeation through lipid alone. The initial rate of passage exceeds the rate of unhindered passage (no lipid film) through the same cross sectional area by 10 fold.