Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum

Abstract

The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (⌬) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H ؉ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H ؉ -arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.Amino acids are utilized by fungi as primary or secondary nitrogen sources or as building blocks for the synthesis of proteins and peptides. Systems involved in the translocation of amino acids across the plasma membrane have been studied in only a few filamentous fungi (5,8,25). Of these fungi, Neurospora crassa, Aspergillus nidulans, and, to a lesser extent, Penicillium chrysogenum are genetically and biochemically the most extensively characterized species. Two distinct classes of plasma membrane-located amino acid permeases are found in filamentous fungi: systems that catalyze the uptake of structurally related amino acids with a broad substrate specificity, such as the general amino acid permeases of plant and animal cells (2,15,18), and systems with a narrow substrate specificity, such as bacterial amino acid transporters (22).Fungi show a peculiar substrate specificity in their amino acid transport systems, and multiple transport mechanisms seem to exist for several amino acids. In N. crassa, five distinct transport systems have been identified, with specificity for aromatic and aliphatic amino acids (system I); aromatic, aliphatic, and basic amino acids (system II); basic amino acids (system III); acidic amino acids (system IV); and L-methionine (system V) (5,8,20,25). Studies with mycelium of P. chrysogenum indicate that this fungus possesses at least six distinct amino acid transport systems (1, 5, 8-10, 11, 24). Most of these systems are specific for one amino acid and analogs, except for system III, which is a general amino acid permease, and system IV, which transports acidic amino acids only (8, 10). Amino acid transporters of fungi are assumed to possess some typical properties: (i) they seem to function unidirectionally; i.e., only uphill transport is observed, while efflux or countertransport of the accumulated amino acids is not detected (8, 19); and (ii) their activity is regulated by transinhibition, i.e., a high internal concentration of an amino acid appears to lower the activity of the transport syst...

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