Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1 Article (Faculty180)

cited authors

  • Bolger, M B; Haworth, I S; Yeung, A K; Ann, D; von Grafenstein, H; Hamm-Alvarez, S; Okamoto, C T; Kim, K J; Basu, S K; Wu, S; Lee, V H


  • The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

publication date

  • 1998

published in

start page

  • 1286

end page

  • 91


  • 87