Moskaug J O, Prydz K, Olsnes S
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):473-7. doi: 10.1042/bj2910473.
We have developed a system that makes it possible to study the fate of a foreign polypeptide that is inserted in the plasma membrane. Diphtheria toxin is a bacterial protein toxin that, upon acidification, has the ability to insert into the plasma membrane from the outside of eukaryotic cells. We present results that indicate endocytic uptake and degradation of the diphtheria toxin B-fragment after insertion into the membrane of Vero cells. The degradation rate of the fragment was found to be very high (t1/2 = 6 min) and dependent on cleavage of the extracellular part of the polypeptide with protease. Degradation was strongly inhibited in ATP-depleted cells, as well as at temperatures below 18 degrees C, and it was partially inhibited when the cytosol was acidified to block endocytosis from clathrin-coated pits. Degradation was also reduced in the presence of NH4Cl. The results indicate that the inserted and cleaved B-fragment is degraded by a process requiring endocytosis and transport to late endosomes or to lysosomes.
我们开发了一种系统，该系统使研究插入质膜的外源多肽的命运成为可能。白喉毒素是一种细菌蛋白毒素，在酸化后能够从真核细胞外部插入质膜。我们展示的结果表明，白喉毒素B片段插入Vero细胞质膜后会发生内吞摄取和降解。发现该片段的降解速率非常高（半衰期 = 6分钟），并且依赖于用蛋白酶切割多肽的细胞外部分。在ATP耗尽的细胞中以及在低于18摄氏度的温度下，降解受到强烈抑制，并且当细胞质酸化以阻断从网格蛋白包被小窝的内吞作用时，降解受到部分抑制。在存在NH4Cl的情况下，降解也会减少。结果表明，插入并切割后的B片段通过需要内吞作用并转运至晚期内体或溶酶体的过程进行降解。
