TY - JOUR
T1 - The transmembrane tyrosines Y56, Y91 and Y167 play important roles in determining the affinity and transport rate of the rabbit proton-coupled peptide transporter PepT1
AU - Pieri, Myrtani
AU - Gan, Christine
AU - Bailey, Patrick
AU - Meredith, David
PY - 2009/11
Y1 - 2009/11
N2 - The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using site-directed mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots. All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wild-type PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1. Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100-fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.
AB - The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using site-directed mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots. All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wild-type PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1. Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100-fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.
KW - Epithelia
KW - Membrane transport
KW - Nutrient absorption
KW - Protein structure-function
KW - Site-directed mutagenesis
KW - SLC15a1
UR - http://www.scopus.com/inward/record.url?scp=70349267549&partnerID=8YFLogxK
U2 - 10.1016/j.biocel.2009.04.014
DO - 10.1016/j.biocel.2009.04.014
M3 - Article
C2 - 19389486
AN - SCOPUS:70349267549
SN - 1357-2725
VL - 41
SP - 2204
EP - 2213
JO - International Journal of Biochemistry and Cell Biology
JF - International Journal of Biochemistry and Cell Biology
IS - 11
ER -