Investigation of the Endoplasmic Reticulum Localization of UDP-Glucuronosyltransferase 2B7 with Systematic Deletion Mutants

Abstract

UDP-Glucuronosyltransferase (UGT) plays an important role in the metabolism of endogenous and exogenous compounds. UGT is a type I membrane protein, and has a dilysine motif (KKXX/KXKXX) in its C-terminal cytoplasmic domain. Although a dilysine motif is defined as an endoplasmic reticulum (ER) retrieval signal, it remains a matter of debate whether this motif functions in the ER localization of UGT. To address this issue, we generated systematic deletion mutants of UGT2B7, a major human isoform, and compared their subcellular localizations with that of an ER marker protein calnexin (CNX), using subcellular fractionation and immunofluorescent microscopy. We found that although the dilysine motif functioned as the ER retention signal in a chimera that replaced the cytoplasmic domain of CD4 with that of UGT2B7, UGT2B7 truncated mutants lacking this motif extensively colocalized with CNX, indicating dilysine motif-independent ER retention of UGT2B7. Moreover, deletion of the C-terminal transmembrane and cytoplasmic domains did not affect ER localization of UGT2B7, suggesting that the signal necessary for ER retention of UGT2B7 is present in its luminal domain. Serial deletions of the luminal domain, however, did not affect the ER retention of the mutants. Further, a cytoplasmic and transmembrane domain-deleted mutant of UGT2B7 was localized to the ER without being secreted. These results suggest that UGT2B7 could localize to the ER without any retention signal, and lead to the conclusion that the static localization of UGT results from lack of a signal for export from the ER.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

“…Therefore, direct interactions between the catalytic domains of UGTs and the heme domains of P450s do not seem possible. However, the transmembrane helix of UGT is known to pass through the membrane and expose a C-terminal portion of the protein of about 20 amino acids to the cytoplasmic side of the ER [ 59 , 60 ]. The studies on the interactions of CYP3A4 with UGT2B7 suggest that the contacting loci of the two proteins involve the cytosolic fragment of UGT [ 61 , 62 ] and the α-helix J of CYP3A4 [ 63 ].…”

Advantage of a Co-expression System for Estimating Physiological Effects of Functional Interaction Between Cytochrome P450 3A4 and Uridine 5’-Diphospho-Glucuronosyltransferase 2B7

“…This domain was believed to work in ER targeting, but our recent study indicated that UGT2B7 can be retained in the ER in a domainindependent fashion. 22) We also determined that the luminal membrane binding site of UGT2B7 is necessary for the interaction with CYP3A4, and that this P450-UGT interaction occurs through the ER membrane. 10) Since it is unlikely that the luminal domain of UGT2B7 associates with CYP3A4, which is in a different membrane, in the absence of detergent, only the cytoplasmic domain of UGT2B7 can associate with CYP3A4 in the simple mixing of microsomes method (Fig.…”

Integrate thermostabilized fusion protein apocytochrome b562RIL and N-glycosylation mutations: A novel approach to heterologous expression of human UDP-glucuronosyltransferase (UGT) 2B7

“…It is suggested that deletions of the C-terminal transmembrane helix (TH) and di-lysine motif (DM, residues 524-529) have no impact on the UGT2B7 location in ER (Miyauchi et al, 2019). Besides, the CTDs of UGTs are conserved, and the fused His-tag to the UGTs C-terminus have no impact on the glucuronidation activity (Zhang et al, 2012b).…”