EXPRESSION OF THE MAMMALIAN RENAL PEPTIDE TRANSPORTER PEPT2 IN THE YEAST PICHIA-PASTORIS AND APPLICATIONS OF THE YEAST SYSTEM FOR FUNCTIONAL-ANALYSIS

It has recently been identified that PEPT2 cDNA encodes the high affin ity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 represents a 729 amino acid protein with 12 putative transmembrane dom ains that mediates H+/H3O+ dependent electrogenic transmembrane transp ort of di- and tripeptides and of selected peptidomimetics. Here the f unctional expression of PEPT2 in the methylotropic yeast Pichia pastor is is described under the control of a methanol inducible promoter. We stern blot analysis of Pichia cell membranes prepared from a recombina nt clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 ti mes higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dip eptide H-3-D-Phe-Ala with an app. K-0.5 Of 0.143 +/- 0.016 mM. Inhibit ion of H-3-D-PheAla uptake by selected di- and tripeptides and beta-la ctam antibiotics revealed the same substrate specificity as obtained I n renal membrane vesicles or for PEPT2 when expressed in Xenopus laevi s oocytes. A novel fluorescence based assay for assessing transport fu nction based on a coumarin-labeled fluorescent peptide analogue has al so been developed. Moreover, using a histidyl auxotrophe strain a PEPT 2 expressing cell clone in which transport function can be monitored b y a simple yeast growth test was established. In conclusion, this is o ne of only a few reports on successful functional expression of mammal ian membrane transport proteins in yeast. The high expression level wi ll provide a simple means for future studies either on the structure-a ffinity relationship for substrate interaction with PEPT2 or for selec tion of mutants generated by random mutagenesis.