Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events
Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events
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DOI：
10.1096/fasebj.14.5.769
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发表时间：
2000-04-01
期刊：
影响因子：
4.8
通讯作者：
Sitia, R
中科院分区：
文献类型：
--
作者：
Mancini, R;Fagioli, C;Sitia, R
许多在内质网（ER）中合成的异常或未组装的蛋白质被胞质蛋白酶体降解。为了研究可溶性糖蛋白的降解是如何被逆转运穿过内质网膜，我们分析了两个IgM亚基，μ和J，保留在内质网的骨髓瘤细胞，不合成轻链的命运。蛋白酶体抑制剂可防止mu和J的降解，这表明两条链都被蛋白酶体逆转位处理。事实上，当蛋白酶体被抑制时，一些不再含有链内二硫键的去糖基化J链在胞质溶胶中积累。然而，丰富的糖基化的J链仍然存在于ER中的时间点，其中降解将已几乎完全在蛋白酶体抑制剂的情况下，这表明逆易位和降解耦合事件。蛋白酶保护和细胞分级分离试验证实了这一点，这表明当蛋白酶体被抑制时，几乎所有的μ链都以糖基化状态保留在ER腔中。与钙连接蛋白的关联与μ链移位到胞质溶胶的失败相关。两者合计，这些结果表明，活性蛋白酶体是必需的提取IG亚基从ER，虽然要求retrotranslocation可能会有所不同，个别基板。
Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu, and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.