Locker J K, Klumperman J, Oorschot V, Horzinek M C, Geuze H J, Rottier P J
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands.
J Biol Chem. 1994 Nov 11;269(45):28263-9.
The M protein of mouse hepatitis virus (MHV) is a triple-spanning membrane glycoprotein that is exclusively O-glycosylated. When expressed independently, it accumulates in late Golgi and the trans-Golgi network (TGN) (Locker, J. K., Griffiths, G., Horzinek, M. C., and Rottier, P. J. M. (1992) (J. Biol. Chem. 267, 14094-14101). To analyze the domains of this protein responsible for its localization, we have generated deletion mutants by site-directed mutagenesis and analyzed their intracellular transport. The intracellular distribution of the mutant proteins was determined by following the extent of O-glycosylation in pulse-chase experiments, by electron microscopic immunocytochemistry, and by surface immunoprecipitation. Mutant proteins lacking the first or the first and second transmembrane domains were not efficiently retained in the Golgi complex or TGN. The latter mutant proteins also localized to endocytic compartments but were not subject to rapid lysosomal degradation. Deletion of the COOH-terminal 22 amino acids, including a tyrosine residue in the context of a potential internalization signal, resulted in plasma membrane exposure of the respective mutant protein. We show that the wild-type MHV-M protein does not recycle between the plasma membrane and the TGN, but rather behaves as a late Golgi/TGN resident in our assays. We propose that the MHV-M protein is retained in the Golgi by two signals, one contained in the cytoplasmic tail and the other determined by the transmembrane domains.
小鼠肝炎病毒（MHV）的M蛋白是一种仅进行O-糖基化修饰的三跨膜糖蛋白。当单独表达时，它会在高尔基体晚期和反式高尔基体网络（TGN）中积累（Locker, J. K., Griffiths, G., Horzinek, M. C., and Rottier, P. J. M. (1992) (J. Biol. Chem. 267, 14094 - 14101)）。为了分析该蛋白中负责其定位的结构域，我们通过定点诱变产生了缺失突变体，并分析了它们的细胞内运输过程。通过脉冲追踪实验中O-糖基化的程度、电子显微镜免疫细胞化学以及表面免疫沉淀来确定突变蛋白的细胞内分布。缺少第一个或第一个和第二个跨膜结构域的突变蛋白不能有效地保留在高尔基体复合体或TGN中。后一种突变蛋白也定位于内吞小室，但不会迅速被溶酶体降解。缺失包括潜在内化信号中的一个酪氨酸残基在内的COOH末端22个氨基酸，导致相应的突变蛋白暴露于质膜。我们发现野生型MHV-M蛋白不会在质膜和TGN之间循环，而是在我们的实验中表现为高尔基体晚期/TGN驻留蛋白。我们提出MHV-M蛋白通过两个信号保留在高尔基体中，一个信号包含在细胞质尾巴中，另一个信号由跨膜结构域决定。
