Specificity and regulation of methionine transport in filamentous fungi

Abstract

l-Methionine uptake by mycelium of Penicillium chrysogenum (under physiological conditions) is mediated by two distinctly different, independently regulated, stereospecific membrane transport systems (permeases). One of the systems is relatively specific for l-methionine. The other is a general (nonspecific) amino acid permease. The specific l-methionine permease develops upon sulfur-starvation. l-methionine transport by sulfur-starved mycelium is at least ten times more rapid than by sulfur-sufficient mycelium at low external methionine concentrations and is strongly inhibited only by analogs and derivatives of l-methionine (e.g., l-ethionine, ethionine and methionine sulfoxides, sulfones, sulfoximines, methyl and ethyl esters, and homocysteine). Nonsulfur amino acids show little or no inhibition. Sulfur-starvation has little effect on the rates of transport of nonsulfur amino acids. The specific system is (a) pH-dependent (optimum at pH 6 with 50% of maximum velocity at pH 3 and 9), (b) temperature-dependent ( Q 10 of 2 between 5 ° and 35 °), (c) energy-dependent (inhibited by 2,4-dinitrophenol, azide, and p-chloromercuribenzoate), (d) relatively insensitive to variations in the ionic strength of the incubation medium, (e) independent of metal ions for activity, and (f) independent of concurrent protein synthesis. Transport is concentration-dependent showing saturation kinetics. The V max in sulfur-sufficient and sulfur-deficient mycelia is 1–3 μmoles/gm-minute. Sulfur starvation results in a decrease in the K m for l-methionine from 10 −3 to 10 −5 m. Refeeding sulfur-starved mycelia with various sulfur compounds results in a rapid increase in the K m back to the sulfur-sufficient level. These results suggest that the specific l-methionine permease is regulated primarily by feedback inhibition-deinhibition rather than by repression-derepression. Inhibition studies suggest that the minimum structural requirements for strong interaction of a compound with the specific methionine permease include (a) a sulfur atom containing no ionizable groups, (b) two methylene groups between the sulfur atom and the α-carbon, (c) an unsubstituted l-α-amino group, (d) an unsubstituted α-hydrogen, and (e) a primary carbonyl group. The nonspecific transport system is deinhibited, or derepressed (or both), upon nitrogen starvation. l-methionine transport by nitrogen-deficient mycelia is 20–100 times more rapid than by nitrogen-sufficient mycelia at low external methionine concentrations, and is strongly inhibited by almost all neutral and basic l-α-amino acids. Nitrogen starvation results in an increase in the rates of transport of all other amino acids tested ( l-tryptophan, l-phenylalanine, l-leucine, l-serine, l-glutamic acid, and l-lysine).