Kain S R, Platt E J, Brown K S, Black N, Firestone G L
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Biol Chem. 1992 Apr 25;267(12):8128-35.
We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.
我们之前已经表明，糖皮质激素可调节病毒感染的M1.54大鼠肝癌细胞中小鼠乳腺肿瘤病毒（MMTV）蛋白的运输和加工。为了研究细胞内膜完整性对MMTV蛋白成熟的作用，使用布雷菲德菌素A（BFA）破坏内质网和高尔基体之间的膜流。免疫沉淀和免疫荧光显微镜检查显示，在地塞米松存在的情况下，BFA抑制了MMTV糖蛋白的蛋白水解加工、细胞表面递送和外化。糖苷酶消化和蛋白质糖基化抑制剂证实，观察到的MMTV糖蛋白产物表观大小的差异是由于BFA诱导的寡糖加工变化所致。BFA处理抑制了MMTV磷蛋白前体的蛋白水解加工，该前体通常与细胞内膜的细胞质面相关联。盐提取效率的相似性表明，BFA不影响未切割的磷酸化前体的膜亲和力。在一种互补方法中，在用编码正常磷酸化前体但表达缺失其跨膜结构域和细胞质尾巴的截短MMTV糖蛋白的突变MMTV前病毒转染的糖皮质激素处理的HTC细胞中，磷酸化多蛋白的蛋白水解加工未发生。这些结果表明，MMTV糖蛋白和磷蛋白可能在运输途径的后期以它们响应糖皮质激素相互加工所需的方式相互作用，并确立了与细胞内膜的功能相互作用对细胞质MMTV磷蛋白成熟的重要性。
