Intracellular localization of UDP-glucuronosyltransferase expressed from the transfected cDNA in cultured cells.
Liver UDP-glucuronosyltransferase (UDPGT) is localized in the endoplasmic reticulum (ER), with its catalytic domain exposed to the lumenal side of the membrane structure. The proteins expressed from the transfected UDPGT cDNA in cultured cells were found to be localized in the ER membrane. Its enzyme activity was in a latent state and was fully expressed in an in vitro assay system when the membrane integrity was disrupted by a detergent, Triton X-100, suggesting that the orientation of the expressed enzyme in the membrane was the same as that of the liver enzyme. To investigate how the expressed UDPGT was retained in the ER, we constructed chimeric plasmids of cDNAs of UDPGT and ErbB2 which is a receptor protein localized in the cell membranes. Analysis of chimeric proteins expressed in the stable transformants of the cultured cells transfected with these plasmids to reveal that the cytoplasmic tail of UDPGT is responsible for the ER retention of the expressed proteins. Deletion and mutation analysis in the cytoplasmic tail of the enzyme demonstrated that the two lysine residues positioned at 3 and 5 from the C-terminus of the molecule are important for conferring the ER residency. Furthermore, the distance of the ER retention signal composed of the two lysine residues from the transmembrane domain may be influential for the efficiency of the ER retention activity.
Kinosaki
M
M
Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.
Masuko
T
T
Sogawa
K
K
Iyanagi
T
T
Yamamoto
T
T
Hashimoto
Y
Y
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D014162:Transfection
D004247:DNA; D014453:Glucuronosyltransferase