IDENTIFICATION OF THE HISTIDYL RESIDUE OBLIGATORY FOR THE CATALYTIC ACTIVITY OF THE HUMAN H+ PEPTIDE COTRANSPORTERS PEPT1 AND PEPT2/

Histidyl residues are known to be essential for the catalytic function of the H+-coupled peptide transporters expressed in the intestine and the kidney, most likely participating in the binding and translocatio n of Hf. Three histidyl residues are conserved among the intestinal an d renal peptide transporters (PEPT1 and PEPT2, respectively) from diff erent animal species, In hPEPT1, these residues are His-57, His-121, a nd His-260. The corresponding residues in hPEPT2 are His-87, His-142, and His-278. We have individually mutated each of these histidyl resid ues in hPEPT1 and in hPEPT2 and compared the catalytic function of the mutants with that of their respective wild type transporters by expre ssing the transporters in Xenopus laevis oocytes and also in HeLa cell s, His-57 in hPEPT1 and His-87 in hPEPT2 were found to be absolutely e ssential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-121 in hPEPT1 is not ess ential since mutation of this residue did not impair transport functio n, His-142 in hPEPT2 was found to play a significant role in the maint enance of transport function but was not found to be obligatory becaus e the mutant had appreciable transport activity. The obligatory histid yl residue (His-57 in hPEPT1 and His-87 in hPEPT2) is located in an al most identical topological position in both transporters, near the ext racellular surface of the second putative transmembrane domain. The se cond conserved histidyl residue is located in the fourth putative tran smembrane domain in hPEPT1 as well as in hPEPT2. The third conserved h istidyl residue is present in the cytoplasmic loop between the transme mbrane domains 6 and 7 and is unlikely to play any significant role in the binding and translocation of H+ and this was supported by the fin dings that mutation of this histidyl residue in hPEPT1 did not interfe re with transport function. The loss of transport function of hPEPT1 a nd hPEPT2, when His-57 in hPEPT1 and His-87 in hPEPT2 were mutated, wa s not due to alterations in protein expression because the expression levels of these mutants were similar to those of the respective wild t ype transporters in HeLa cells as assessed by immunoblot analysis. Con focal analysis of immunofluorescence in X. laevis oocytes expressing t he wild type and the three histidine mutants of hPEPT1 showed that the transporter protein is expressed exclusively in the plasma membrane a nd that the level of expression is comparable among the wild type and the three mutants. These site-directed mutagenesis studies clearly sho w that His-57 in hPEPT1 and His-87 in hPEPT2 an the most critical hist idyl residues necessary for the catalytic function of these transporte rs.