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Evidence that the rabbit proton-peptide co-transporter PepT1 is a multimer when expressed in Xenopus laevis oocytes

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Abstract

To test whether the rabbit proton-coupled peptide transporter PepT1 is a multimer, we have employed a combination of transport assays, luminometry and site-directed mutagenesis. A functional epitope-tagged PepT1 construct (PepT1-FLAG) was co-expressed in Xenopus laevis oocytes with a non-functional but normally trafficked mutant form of the same transporter (W294F-PepT1). The amount of PepT1-FLAG cRNA injected into the oocytes was kept constant, while the amount of W294F-PepT1 cRNA was increased over the mole fraction range of 0 to 1. The uptake of [3H]-d-Phe-l-Gln into the oocytes was measured at pHout 5.5, and the surface expression of PepT1-FLAG was quantified by luminometry. As the mole fraction of injected W294F-PepT1 increased, the uptake of d-Phe-l-Gln decreased. This occurred despite the surface expression of PepT1-FLAG remaining constant, and so we can conclude that PepT1 must be a multimer. Assuming that PepT1 acts as a homomultimer, the best fit for the modelling suggests that PepT1 could be a tetramer, with a minimum requirement of two functional subunits in each protein complex. Western blotting also showed the presence of higher-order complexes of PepT1-FLAG in oocyte membranes. It should be noted that we cannot formally exclude the possibility that PepT1 interacts with unidentified Xenopus protein(s). The finding that PepT1 is a multimer has important implications for the molecular modelling of this protein.

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Acknowledgement

We thank the Wellcome Trust for generously funding this work and for a Prize Studentship to KEP. We also wish to thank Professor C. Korbmacher (University of Erlangen, Germany) for advice and assistance with the luminometry.

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Correspondence to David Meredith.

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Panitsas, KE., Boyd, C.A.R. & Meredith, D. Evidence that the rabbit proton-peptide co-transporter PepT1 is a multimer when expressed in Xenopus laevis oocytes. Pflugers Arch - Eur J Physiol 452, 53–63 (2006). https://doi.org/10.1007/s00424-005-0002-0

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  • DOI: https://doi.org/10.1007/s00424-005-0002-0

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