Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis

Abstract

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides

“…The accumulation of the amino acids present in the peptides was clearly not due to external breakdown of the peptides followed by transport of the amino acids since (i) no free amino acids were detected in the external medium, (ii) the accumulation of amino acids was observed only with transportable peptides, the nontransported peptides were hydrolyzed upon permeabilization of the cells, and (iii) the di-and tripeptide transport activity was completely lost in the ⌬dtpT FCA r mutant, the peptidase activities in the wildtype strain and in the ⌬dtpT FCA r mutant were similar. DtpT and the newly described DtpP system have overlapping specificities for di-and tripeptides but do not transport amino acids or oligopeptides (Table 2 and results not shown) (11,25,26). The DtpT carrier transports relatively hydrophilic di-and tripeptides such as Glu-Val, Gly-Asp, Met-Asp, Ala-Gly, and Pro-Gly (6,26; also this study), whereas DtpP transports a variety of structurally different di-and tripeptides, with an apparent preference for hydrophobic (branched-chain) residues.…”

“…The Opp system of L. lactis also belongs to this family of transporters (29). On the contrary, the di-and tripeptide carrier (DtpT) of L. lactis is a secondary transport system which uses the proton motive force to accumulate peptides (6,25). The DtpP system described in this study resembles the ATP-dependent peptide transporter of E. coli and S. typhimurium as well as the Opp system of L. lactis with regard to energy coupling to transport.…”

Utilisation des sources d'azote du lait par Lactococcus lactis

“…Le système de transport DtpT a été le premier caractérisé chez L laetis (Smid et al, 1989 ;Kunji et al, 1993). Il transporte exclusivement des di-et tripeptides.…”

Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis

“…One uptake system specific for di-and tripeptides has been investigated in a peptidase-free membrane vesicle system (23). In that study, alanyl-glutamate (Ala-Glu) was used as a model substrate.…”

“…Accumulation of the dipeptide in membrane vesicles of Lactococcus lactis ML3 fused with liposomes containing beef heart cytochrome c oxidase was found to be driven by the electrical potential (A+f) and the chemical gradient of protons (ApH) across the membrane (23). Information about the specificity of this transport system is limited and is restricted to results obtained from competition experiments in which the rate of uptake of radioactively labelled peptides was estimated in the absence or presence of a few unlabelled peptides (10,22,23 in-derived amino acids will be supplied to the cells as proline-containing peptides. The finding that especially proline-containing dipeptides are high-affinity substrates for the lactococcal di-tripeptide transport system is in agreement with this notion (24).…”