Rothman J E, Urbani L J, Brands R
J Cell Biol. 1984 Jul;99(1 Pt 1):248-59. doi: 10.1083/jcb.99.1.248.
Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.
形成了包含水泡性口炎病毒感染的中国仓鼠卵巢克隆15B细胞（缺乏高尔基体酶UDP-N-乙酰葡糖胺转移酶I）和未感染的野生型中国仓鼠卵巢细胞的混合单层。单层暴露于pH 5.0后会发生广泛的细胞融合。融合时存在于15B细胞粗面内质网（用[35S]甲硫氨酸标记）或高尔基体复合体（用[3H]棕榈酸标记）中的水泡性口炎病毒编码膜糖蛋白（G蛋白）在融合后可从野生型细胞到达高尔基体复合体；质膜中的G蛋白则不能。通过G蛋白到达时转化为耐内切糖苷酶H的形式来监测其向野生型高尔基体复合体的转移，免疫荧光显微镜检查也证实了这一点。融合时15B细胞高尔基体复合体中的G蛋白在转移到外源性高尔基体群体方面表现出的特性，与早期在无细胞系统中发现的特性相同（弗里斯，E.，和J.E.罗斯曼。1981年。《细胞生物学杂志》，90: 697 - 704）。具体而言，使用体内融合和体外测定的脉冲追踪实验揭示了高尔基体复合体中相同的两种G蛋白群体。第一种群体由刚接受脂肪酸的G蛋白分子组成，在体内和体外都能转移到第二种高尔基体群体。第二种群体在G蛋白酰化后约5分钟进入，在体内和体外都无法进行这种转移。据推测，第二种群体由那些在细胞融合或体外测定匀浆之前的等效过程中已经在15B高尔基体群体内各间隔之间转移的G蛋白分子组成。显然，这两种测量方法检测到的是高尔基体复合体中相同的间隔边界。因此，体内发生的糖蛋白在高尔基体堆叠之间出人意料地容易进行的转运过程在体外可能得以保留，为无细胞系统提供了基础。
