Wesche J, Rapak A, Olsnes S
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
J Biol Chem. 1999 Nov 26;274(48):34443-9. doi: 10.1074/jbc.274.48.34443.
Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.
蓖麻毒素的作用机制是将具有酶活性的毒素A链转运至胞质溶胶，该毒素会使核糖体失活。在改变细胞对毒素敏感性的条件下，研究了蓖麻毒素的逆行细胞内运输和转运过程。为此，监测了高尔基体中突变A链的酪氨酸硫酸化、内质网（ER）中的糖基化以及胞质部分中A链的出现情况。将内质网回收信号（C末端KDEL序列）引入A链会增加毒性，并导致更有效的糖基化，这表明从高尔基体到内质网的运输增强。钙耗竭既不抑制硫酸化也不抑制糖基化，但会抑制转运和毒性，这表明毒素是通过靶向蛋白酶体进行降解的错误折叠蛋白所使用的途径转运至胞质溶胶的。微酸化培养基有类似的效果。蛋白酶体抑制剂乳胞素会使细胞对蓖麻毒素敏感，并增加胞质溶胶中蓖麻毒素A链的含量。抗Sec61α沉淀出硫酸化和糖基化的蓖麻毒素A链，这表明毒素的逆行转运涉及Sec61p。数据表明，穿过内质网膜的逆行转运是中毒所必需的。
