Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences (Y.M., A.K., K.K., Y.H., K.F., Y.T.) and Laboratory of Molecular Life Sciences, Graduate School of Pharmaceutical Sciences (Y.M., S.K., Y.I.), Kyushu University, Fukuoka, Japan; and Department of Clinical Pharmacology, Flinders Medical Centre and Flinders University, Adelaide, South Australia, Australia (P.I.M.)
Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences (Y.M., A.K., K.K., Y.H., K.F., Y.T.) and Laboratory of Molecular Life Sciences, Graduate School of Pharmaceutical Sciences (Y.M., S.K., Y.I.), Kyushu University, Fukuoka, Japan; and Department of Clinical Pharmacology, Flinders Medical Centre and Flinders University, Adelaide, South Australia, Australia (P.I.M.).
Mol Pharmacol. 2019 May;95(5):551-562. doi: 10.1124/mol.118.113902. Epub 2019 Apr 3.
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.
UDP-葡萄糖醛酸基转移酶 (UGT) 在内源性和外源性化合物的代谢中发挥重要作用。UGT 是一种 I 型膜蛋白，其胞质域 C 末端具有双赖氨酰基基序 (KKXX/KXKXX)。虽然双赖氨酰基基序被定义为内质网 (ER) 回收信号，但该基序是否在 UGT 的 ER 定位中起作用仍存在争议。为了解决这个问题，我们生成了人主要同工酶 UGT2B7 的系统缺失突变体，并通过亚细胞分级分离和免疫荧光显微镜比较了它们与 ER 标记蛋白钙连蛋白 (CNX) 的亚细胞定位。我们发现，尽管双赖氨酰基基序在取代 CD4 胞质域的 UGT2B7 嵌合体中起 ER 保留信号的作用，但缺乏该基序的 UGT2B7 截断突变体与 CNX 广泛共定位，表明 UGT2B7 存在不依赖于双赖氨酰基基序的 ER 保留。此外，缺失 C 端跨膜和胞质域不影响 UGT2B7 的 ER 定位，表明 UGT2B7 用于 ER 保留的信号存在于其腔域中。然而，腔域的串联缺失不影响突变体的 ER 保留。此外，UGT2B7 的胞质和跨膜结构域缺失突变体在不被分泌的情况下被定位到 ER。这些结果表明，UGT2B7 可以在没有任何保留信号的情况下定位到 ER，并且得出结论，UGT 的静态定位是由于缺乏从 ER 输出的信号所致。
