UDP-glucuronosyltransferase 1A9 function is mediated by the two C-terminal lysine residues and proper mobility/flexibility of the cytoplasmic tail

UDP-glucuronosyltransferase (UGT) is a type-I membrane protein that is localized to the endoplasmic reticulum (ER). The main body of the UGT is located on the luminal side of the ER, whereas a single transmembrane helix, followed by the cytoplasmic tail, is at its C-terminus. At the end of the cytoplasmic tail, UGT has a di-lysine motif (KSKTH in UGT1A9), which is originally defined as an ER-targeting peptide. However, we previously revealed that this motif is important for maintaining enzyme activity, rather than ER localization in UGT1A9. To elucidate the molecular mechanism underlying the cytoplasmic tail-mediated maintenance of UGT1A9 activity, additional mutants were generated and their activities were measured. Introducing single amino acid substitutions at the third or fifth lysine residue from the C-terminus decreased the enzymatic activity of UGT1A9 with substitution of the third residue resulting in greater suppression. Enzyme activity was markedly reduced in the mutant with a deleted cytoplasmic tail, indicating that the mobility/flexibility of the cytoplasmic tail, in addition to the two C-terminal residues, is necessary for UGT1A9 activity. The cytoplasmic tail does not directly affect the main body of the enzyme and two molecular mechanisms for maintaining UGT1A9 activity are proposed.