Expression of the mammalian renal peptide transporter PEPT2 in the yeastPichia pastorisand applications of the yeast system for functional analysis

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Abstract
It has recently been identified that PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPTP represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di -and tripeptides and of selected peptidomimetics. Here the functional expression of PEPTP in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85–87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app. K05 of 0.143 ± 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di -and tripeptides and β-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPTP when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a cournarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPTP expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPTS or for selection of mutants generated by random mutagenesis.