Uhlin-Hansen L, Yanagishita M
Institute of Medical Biology, University of Tromsø, Norway.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):271-8. doi: 10.1042/bj3100271.
Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.
用[35S]硫酸盐标记大鼠卵巢颗粒细胞0.5 - 20小时，并在存在或不存在1 - 2微克/毫升布雷菲德菌素A（BFA）的情况下追踪长达21小时。然后通过凝胶色谱分析来自培养基、质膜和细胞内部分的硫酸乙酰肝素[35S]硫酸盐（HS）蛋白聚糖。在不存在BFA的情况下，约85%与质膜相关的HS蛋白聚糖被内吞，随后在细胞内降解。未观察到HS蛋白聚糖在细胞内池和细胞表面之间的再循环。将细胞暴露于BFA少于1小时不会影响HS蛋白聚糖的周转，而该药物对高尔基体功能的影响在约10分钟时达到最大值。当细胞用BFA处理超过1 - 2小时时，HS蛋白聚糖的内吞率降低至对照的约50%。药物不影响内吞的HS蛋白聚糖向溶酶体的递送。环己酰亚胺也降低了HS蛋白聚糖的内吞，但程度不如BFA，这表明BFA的抑制作用只能部分归因于内质网到质膜的蛋白质转运受阻。与HS蛋白聚糖的内吞不同，已知由网格蛋白包被小泡介导的125I - 转铁蛋白的内吞以及作为批量内吞标记分子的125I - 蓖麻毒素的内吞均不受BFA影响。质膜中125I - 转铁蛋白和125I - 蓖麻毒素的半衰期分别约为10分钟和25分钟，而HS蛋白聚糖约为5小时。总之，这些结果表明，与质膜相关的HS蛋白聚糖的内吞是由与大多数其他细胞表面蛋白内吞不同的机制介导的。此外，HS蛋白聚糖内吞所涉及的机制对BFA敏感。
