The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal.
Microsomal-type cytochrome P450s are integral membrane proteins bound to the membrane through their N-terminal transmembrane hydrophobic segment, the signal anchor sequence. To elucidate the determinants that enable the P450s to be located in the ER, we constructed cDNAs encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase Sec, was connected to the N-terminus of the full-length or truncated forms of a microsomal-type P450, P450(M1), and the constructed plasmids were expressed in COS cells. Since carboxyesterase Sec is an N-glycosylated secretory protein, endo H treatment could be used to determine whether these chimeric proteins were located in the ER or not. Carboxyesterase Sec with the N-terminal 20 amino acids, containing the transmembrane region, of P450(M1), was located in the ER, as determined from the endo H sensitivity of the expressed protein and immunofluorescence staining of the cells. As the expressed protein exhibited carboxyesterase activity, it was not retained in the ER through the BiP-dependent quality control system recognizing unfolded proteins. Another chimeric protein construct in which carboxyesterase Sec was connected to the C-terminal region of rat UDP-glucuronosyltransferase (UDP-GT), that contained a double-lysin ER retention motif, was also located in the ER, as determined from the endo H sensitivity and immunofluorescence staining. On the other hand, the sugar moiety of the carboxyesterase Sec connected to the transmembrane segment of UDP-GT, Sec/GTd, was partially resistant to the endo H treatment. From the results of immunofluorescent staining and cell fractionation, it was concluded that the Sec/GTd product was located in the Golgi apparatus. These observations indicated that the N-terminal hydrophobic segment of P450(M1) is sufficient for the ER membrane retention, whereas the transmembrane segment of UDP-GT is not. To determine whether microsomal P450s are recycled between the ER and Golgi compartments or not, a DNA construct encoding cathepsin D connected to the N-terminus of P450(M1) was prepared and expressed in COS cells. The fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. As a control, authentic cathepsin D was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. Since GlcNAc-P-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the Golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin D portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)
Murakami
K
K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Mihara
K
K
Omura
T
T
D000818:Animals; D001483:Base Sequence; D002402:Cathepsin D / Q000235:genetics; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D002402:Cathepsin D