Ono A, Kawakita M
Department of Physiological Chemistry, Tokyo Metropolitan Institute of Medical Science, Rinshoken.
J Biochem. 1995 Dec;118(6):1248-54. doi: 10.1093/oxfordjournals.jbchem.a125014.
Thapsigargin and ionomycin inhibited the intracellular transport of the Sendai virus F0 protein. Depletion of Ca2+ from intracellular Ca2+ store(s) is critical for the inhibition, since ionomycin was more effective in the absence of extracellular Ca2+ than in its presence. Transport of F0 was arrested between the trans-Golgi complex and the plasma membrane [Ono, A. and Kawakita, M. (1994) J. Biochem. 116, 649-656], but only when thapsigargin was added before the synthesis of F0. This implies that F0 was committed to later arrest in the endoplasmic reticulum. Non-reducing SDS-polyacrylamide gel electrophoresis revealed a conformational abnormality of F0 immediately after pulse-labeling in thapsigargin-treated cells. The abnormality did not affect the exit of F0 from the endoplasmic reticulum, but paralleled its later arrest at the trans-Golgi stage. Pulse-labeled and 1-h-chased F0 was endoglycosidase H-resistant even in thapsigargin-treated cells, but was not recognized by mAb f-49, a monoclonal antibody that recognizes the corresponding F0 intermediate in uninhibited cells. The misfolded F0 may escape from recognition by means of the quality control system of the endoplasmic reticulum, but another system in the Golgi complex may complement the former. The arrested F0 was rapidly degraded.
毒胡萝卜素和离子霉素抑制了仙台病毒F0蛋白的细胞内运输。细胞内钙库中Ca2+的耗尽对于这种抑制作用至关重要，因为离子霉素在无细胞外Ca2+时比有细胞外Ca2+时更有效。F0的运输在反式高尔基体复合体和质膜之间被阻断[小野，A.和川北，M.（1994年）《生物化学杂志》116，649 - 656]，但只有当在F0合成之前加入毒胡萝卜素时才会如此。这意味着F0注定会在稍后在内质网中被阻断。非还原SDS - 聚丙烯酰胺凝胶电泳显示，在毒胡萝卜素处理的细胞中脉冲标记后，F0立即出现构象异常。这种异常并不影响F0从内质网的输出，但与它稍后在反式高尔基体阶段的阻断情况平行。即使在毒胡萝卜素处理的细胞中，脉冲标记并经过1小时追踪的F0对内切糖苷酶H具有抗性，但不能被单克隆抗体f - 49识别，f - 49是一种能识别未受抑制细胞中相应F0中间体的单克隆抗体。错误折叠的F0可能通过内质网的质量控制系统逃避识别，但高尔基体复合体中的另一个系统可能会补充前者。被阻断的F0会迅速降解。
