EARLY AND LATE FUNCTIONS ASSOCIATED WITH THE GOLGI-APPARATUS RESIDE IN DISTINCT COMPARTMENTS
EARLY AND LATE FUNCTIONS ASSOCIATED WITH THE GOLGI-APPARATUS RESIDE IN DISTINCT COMPARTMENTS
复制标题
DOI：
10.1073/pnas.78.12.7453
复制
发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
ROTHMAN, JE
中科院分区：
文献类型：
--
作者：
DUNPHY, WG;FRIES, E;ROTHMAN, JE
当在蔗糖密度梯度中离心来自中国仓鼠卵巢（CHO）[亲本细胞系，克隆15 B和克隆102]的膜时，催化天冬酰胺连接寡糖的高尔基体相关加工的2个连续阶段的酶分布不同。一种甘露糖苷酶，其仅除去外部α-甘露糖苷酶，将来自水泡性口炎病毒G蛋白的前体寡糖的1，2-连接的甘露糖残基（以产生修剪的寡糖核心）与在糖基化的后期、终末阶段起作用的酶（半乳糖基转移酶和唾液酸转移酶）分离。新鲜酰化的G蛋白与新修剪的寡糖在早期作用膜的分布中结合，由甘露糖苷酶定义;用[3 H]半乳糖脉冲标记的G蛋白分布在晚期作用膜的轮廓中。G蛋白存在于早期作用的膜中的粗馏分可以通过与外源性高尔基体膜在体外孵育的终末糖基化; G蛋白失去了在体外被处理的能力，因为它似乎进入体内的晚期作用的膜。这些实验揭示了存在2个不同的隔室，通过这些隔室，细胞内转运的蛋白质如G按顺序通过高尔基体相关的过程进行。这些隔间可能包括cis和transs两侧的高尔基体堆栈的池。还使用了小鼠成纤维细胞L细胞突变体克隆6。
Enzymes that catalyze the 2 successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary (CHO) [parent cell line, clone 15B, and clone 102] were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, .alpha.-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitits viral G protein (to yield a trimmed oligosaccharide core) was separated from enzymes (galactosyl- and sialyltransferases) that act in the later, terminal stage of glycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase; G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of 2 distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. These compartments probably consist of cisternae on the cis and trans sides of the Golgi stack. Mouse fibroblast L cell mutant clone 6 was also used.