Kinetics and substrate specificity of membrane-reconstituted peptide transporter DtpT of Lactococcus lactis

Abstract

The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[(14)C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, omega-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT(1) and PepT(2) shows that the bacterial transporter is more restrictive in its substrate recognition.

FIG. 1

Kinetic analysis of proton-driven uptake of Pro-[¹⁴C]Ala. Proteoliposomes preloaded with 20 mM KPi, 100 mM KAc, and 2 mM MgSO₄ (pH 6.0) were diluted 50-fold into 120 Na-PIPES, 2 mM MgSO₄, 0.5 μM valinomycin (pH 6.0), plus Pro-[¹⁴C]Ala at concentrations ranging from 0.065 to 10 mM. The inset shows the Eadie-Hofstee transformation of the data; V is the rate of Pro-Ala uptake (in nanomoles per minute per milligram of protein) and S is the millimolar concentration of Pro-Ala in the medium.

FIG. 2

Inhibition of Pro-[¹⁴C]Ala uptake by Ala and Ala-containing peptides. Uptake of Pro-[¹⁴C]Ala (65 μM, final concentration) was determined as described in the legend to Fig. 1, in the absence and presence of increasing concentrations of alanine and alanine-containing peptide series (0.01 to 10 mM). Uptake of Pro-Ala measured in the absence of inhibitors was taken as 100% (46 nmol/min × mg of protein). Symbols: ●, alanine; ○, Ala-Ala; ▾, Ala-Ala-Ala; ▿, Ala-Ala-Ala-Ala; ■, Ala-Ala-Ala-Ala-Ala.

FIG. 3

IC₅₀ values for dipeptides and tripeptides. Uptake of Pro-[¹⁴C]Ala (65 μM, final concentration) was determined as described in the legend to Fig. 1 in the presence of various competing peptides. The IC₅₀ values for different inhibitors were calculated as described in Materials and Methods. The error bars indicate the standard deviations from the means of two independent experiments.

FIG. 4

IC₅₀ values for peptides differing at the amino- or carboxyl-terminal residue. Uptake of Pro-[¹⁴C]Ala (65 μM, final concentration) was determined as described in the legend to Fig. 1 in the presence of various competing peptides. The IC₅₀ values for different inhibitors were calculated as described in Materials and Methods. X corresponds to the variable amino acid at position 1 (A), 2 (B), and 3 (C). The error bars indicate the standard deviations from the means of two independent experiments.

FIG. 5

Structures of phenylalanine-containing peptides and those with a carbobenzoxy group at the first position; the structure of the peptide mimetics 4-amino-butanoic acid and 5-amino-pentanoic acid are also shown.