title: Expression of the mammalian renal peptide transporter PEPT2 in the yeast Pichia pastoris and applications of the yeast system for functional analysis.
abstract: It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 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 PEPT2 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. K0.5 of 0.143 +/- 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 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 PEPT2 or for selection of mutants generated by random mutagenesis.
authors.affiliation: Institute of Nutritional Sciences, University of Giessen, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Döring
authors.forename: T
authors.initials: T
authors.lastname: Michel
authors.forename: A
authors.initials: A
authors.lastname: Rösel
authors.forename: M
authors.initials: M
authors.lastname: Nickolaus
authors.forename: H
authors.initials: H
authors.lastname: Daniel
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011817:Rabbits; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2