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

Pflugers Arch. 2006 Apr;452(1):53-63. doi: 10.1007/s00424-005-0002-0. Epub 2006 Feb 8.

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 [(3)H]-D: -Phe-L: -Gln into the oocytes was measured at pH(out) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Expression
  • Mutation
  • Oocytes / metabolism
  • Peptide Transporter 1
  • Protein Structure, Quaternary
  • RNA, Complementary / metabolism
  • Rabbits
  • Symporters / chemistry*
  • Symporters / metabolism
  • Xenopus laevis

Substances

  • Peptide Transporter 1
  • RNA, Complementary
  • Symporters