Stoichiometry of H +/amino acid cotransport in Neurospora crassa revealed by current-voltage analysis

Abstract

Coupling of ions to the uptake of neutral and basic amino acids via a general amino acid transport system (System II), was studied in a mutant of Neurospora crassa (bat mtr) which lacks other transport systems for these solutes. All amino acids tested — including ones bearing no net charge — elicited rapid membrane depolarization, as expected for ion-coupled transport. (Since amino acid transport in Neurospora is not dependent on extracellular Na + or K +, the associated ion is presumed to be H +.) Although the 14C-labeled amino acid fluxes through System II are largely independent of the identity of the amino acid, the depolarization caused by basic amino acids ( l-lysine and l-ornithine) is 60–70% greater than that for neutral amino acids (e.g. l-leucine). This difference is consistent with a constant H +/amino acid stoichiometry of 2, the extra charge for lysine and ornithine being that on the amino acid itself, so that the charge ratio basic: neutral amino acids is 3:2. When actual membrane charge flow associated with amino acid uptake was compared with measured 14C-labeled amino acid influx, ratios of 2.07 charges/mol l-leucine and 3.40 charges/mol l-lysine were obtained, again in accord with a constant translocation stoichiometry of 2H +/amino acid. The advantages of this electrical method for estimating H +/solute stoichiometry in cotransport are discussed in relation to more familiar methods.