Cytosolic COOH terminus of the peptide transporter PEPT2 is involved in apical membrane localization of the protein.
The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues, including kidney, lung, mammary gland, choroid plexus, and glia cells. In renal tubular cells, PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush-border membrane are not known. We have explored the role of the 36 COOH-terminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wild-type transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK, and the cellular distribution of the fusion proteins was assessed using confocal microscopy. Whereas deletion of the last seven amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids resulted in impaired apical expression and distinct accumulation of mutant proteins in endosomal and lysosomal vesicles. Truncation of more amino acids (delC36) containing tyrosine-based motifs led to a rather diffuse intracellular distribution pattern. Mutations introduced at isoleucine-720 (I720A) and leucine-722 (I722A) also caused an impaired surface appearance. Internalization assays revealed a higher endocytotic rate of the PEPT2 mutants I720A, L722A, and delC36. Our data suggest that a three-amino acid stretch (INL) and tyrosine-based motifs within the COOH tail of PEPT2 are involved in PEPT2's apical membrane localization and membrane steady-state level.
Klapper
Maja
M
Research Group Molecular Nutrition, Univ. of Kiel, Germany.
Daniel
Hannelore
H
Döring
Frank
F
D020816:Amino Acid Motifs; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation; D009893:Opossums; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
D015415:Biomarkers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2