Skretting G, Torgersen M L, van Deurs B, Sandvig K
Institute for Cancer Research at The Norwegian Radium Hospital, Montebello, Norway.
J Cell Sci. 1999 Nov;112 ( Pt 22):3899-909. doi: 10.1242/jcs.112.22.3899.
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.
我们在此使用白喉毒素作为一种工具，来研究糖基磷脂酰肌醇连接分子（一种能够介导中毒的白喉毒素受体的糖基磷脂酰肌醇连接形式）所采用的内吞作用类型。该受体在HeLa细胞中表达，在这些细胞中，网格蛋白依赖性内吞作用可通过突变型发动蛋白的过表达而被阻断。白喉毒素通过首先结合细胞表面受体来使细胞中毒，然后毒素被内吞，并且在暴露于低内涵体pH值时，毒素进入细胞质溶胶，在那里它抑制蛋白质合成。因此，毒素对蛋白质合成的抑制作用可用于探究糖基磷脂酰肌醇连接受体进入酸性区室的情况。此外，毒素的降解可作为进入内涵体/溶酶体区室的指标。数据表明，尽管突变型发动蛋白的表达抑制了经由野生型受体介导的中毒，但突变型发动蛋白并不影响与糖基磷脂酰肌醇连接受体结合的白喉毒素的中毒、内吞作用及降解。共聚焦显微镜检查表明，白喉毒素被转运至含有早期内涵体标志物EEA1的囊泡中。对所使用的HeLa细胞进行的生化和超微结构研究表明，它们的小窝蛋白-1水平非常低，并且在细胞表面几乎没有小窝。此外，与糖基磷脂酰肌醇连接受体结合的白喉毒素的内吞摄取并未因甲基-β-环糊精或制霉菌素而减少，这两种物质都会破坏小窝结构和功能。因此，一种糖基磷脂酰肌醇连接蛋白（在这种情况下是白喉毒素受体）进入内涵体/溶酶体系统的过程可以独立于小窝和网格蛋白包被囊泡而发生。
