Identification of a potential substrate binding domain in the mammalian peptide transporters PEPT1 and PEPT2 using PEPT1-PEPT2 and PEPT2-PEPT1 chimeras.
The mammalian peptide transporters PEPT1 and PEPT2 are energized by a transmembrane electrochemical H+ gradient and exhibit similar broad substrate specificity. These transporters however differ in their affinity for substrates, PEPT1 being a low-affinity transporter and PEPT2 being a high-affinity transporter. To identify the substrate binding domain in PEPT1 and PEPT2 which is responsible for the differing affinities, we constructed a series of PEPT1-PEPT2 and PEPT2-PEPT1 chimeras using an in vivo restriction site-independent procedure and determined their substrate affinities. A comparison of these kinetic data for different chimeras with those of the wild-type PEPT1 and PEPT2 in conjunction with the specific structural PEPT1/PEPT2 crossover regions in these chimeras has led to the identification of a putative substrate binding site, which is comprised of the transmembrane domains 7, 8 and 9 of the transporters.
Fei
Y J
YJ
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Liu
J C
JC
Fujita
T
T
Liang
R
R
Ganapathy
V
V
Leibach
F H
FH
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D009865:Oocytes / Q000378:metabolism; D000074011:Peptide Transporter 1; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D000074011:Peptide Transporter 1; D011993:Recombinant Fusion Proteins; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2