Murakami K, Mihara K, Omura T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
J Biochem. 1994 Jul;116(1):164-75. doi: 10.1093/oxfordjournals.jbchem.a124489.
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)
微粒体型细胞色素P450是通过其N端跨膜疏水片段（信号锚定序列）与膜结合的整合膜蛋白。为了阐明使P450定位于内质网的决定因素，我们构建了编码嵌合蛋白的cDNA，其中分泌型羧酸酯酶（羧酸酯酶Sec）连接到微粒体型P450（P450(M1)）全长或截短形式的N端，构建的质粒在COS细胞中表达。由于羧酸酯酶Sec是一种N-糖基化分泌蛋白，内切糖苷酶H处理可用于确定这些嵌合蛋白是否定位于内质网。从表达蛋白的内切糖苷酶H敏感性和细胞免疫荧光染色确定，含有P450(M1)跨膜区域的N端20个氨基酸的羧酸酯酶Sec定位于内质网。由于表达的蛋白具有羧酸酯酶活性，它不是通过识别未折叠蛋白的BiP依赖性质量控制系统保留在内质网中的。另一种嵌合蛋白构建体，其中羧酸酯酶Sec连接到大鼠UDP-葡萄糖醛酸基转移酶（UDP-GT）的C端区域，该区域含有双赖氨酸内质网保留基序，从内切糖苷酶H敏感性和免疫荧光染色确定也定位于内质网。另一方面，连接到UDP-GT跨膜片段的羧酸酯酶Sec的糖部分，Sec/GTd，对内切糖苷酶H处理有部分抗性。从免疫荧光染色和细胞分级分离结果得出结论，Sec/GTd产物定位于高尔基体。这些观察结果表明，P450(M1)的N端疏水片段足以保留在内质网膜上，而UDP-GT的跨膜片段则不然。为了确定微粒体P450是否在内质网和高尔基体区室之间循环，制备了编码连接到P450(M1) N端的组织蛋白酶D的DNA构建体并在COS细胞中表达。融合蛋白被磷酸化，但磷酸化对碱性磷酸酶敏感。作为对照，对天然组织蛋白酶D的寡糖链进行磷酸化，该磷酸化对碱性磷酸酶处理有抗性。由于在靶向溶酶体蛋白的糖链中形成对碱性磷酸酶抗性磷酸二酯的N-乙酰葡糖胺磷酸转移酶位于高尔基体中，得出结论融合蛋白的组织蛋白酶D部分的寡糖链未被磷酸化，并且嵌合蛋白未进入高尔基体。（摘要截断于400字）
