Evidence that the rabbit proton-peptide co-transporter PepT1 is a multimer when expressed in Xenopus laevis oocytes.
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.
Panitsas
Konstantinos-E
KE
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX, Oxford, UK.
Boyd
C A R
CA
Meredith
David
D
D000818:Animals; D015870:Gene Expression; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D000074011:Peptide Transporter 1; D020836:Protein Structure, Quaternary; D018075:RNA, Complementary / Q000378:metabolism; D011817:Rabbits; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis
D000074011:Peptide Transporter 1; D018075:RNA, Complementary; D027981:Symporters