Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum.
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.
Ouzzine
M
M
UMR-CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, Vandoeuvre-lès-Nancy, France. ouzzine@pharmaco-med.u-nancy.fr
Magdalou
J
J
Burchell
B
B
Fournel-Gigleux
S
S
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D017384:Sequence Deletion
D014453:Glucuronosyltransferase