Expression cloning of a mammalian proton-coupled oligopeptide transporter

Abstract

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1

“…The PEPT1 transporter is predicted to possess 12 transmembrane ~x-helices [16,17,21]. We assumed that histidine residues located in the putative transmembrane ~x-helices play an important role in substrate binding.…”

“…We assumed that histidine residues located in the putative transmembrane ~x-helices play an important role in substrate binding. Seven histidine residues are conserved among rat [21], rabbit [16] and human [17] PEPT1. Among these residues, histidine 57 and histidine 121, which are located in the predicted transmembrane c~-helices 2 and 4, respectively, were of special interest.…”

“…Recently, the H+-coupled oligopeptide transporter PEPT1 has been cloned from rabbit [16] and human [17]. Ganapathy et al [18] reported differential recognition of I]-lactam antibiotics by human PEPT1 and PEPT2, another member of H + / peptide transporter family specifically expressed in the kidney [19].…”

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination

“…Functional expression in X. laevis oocytes was performed as described by Fei et al (1994) with minor modifications. A poly A tail (A 30 ) was inserted between KpnI and SpeI restriction sites of pCRII containing HvPTR1, corresponding to the 3Ј end of the cRNA produced by transcription from the T7 RNA polymerase promoter.…”

A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H + /Sucrose Cotransporter Expressed in Xenopus Oocytes

“…The cotransport of protons with solutes raises the possibility that transport of sucrose by oocytes expressing SUC1 may lead to a decrease in oocyte internal pH, as has been shown for protein/peptide cotransport (Fei et al, 1994). However, we have measured the internal pH of oocytes using proton-selective microelectrodes (Miller et al, 1994).…”