Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity
Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity
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DOI：
10.1091/mbc.11.1.217
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发表时间：
2000-01-01
影响因子：
3.3
通讯作者：
Haas, IG
中科院分区：
文献类型：
--
作者：
Chillarón, J;Haas, IG
小鼠浆细胞瘤细胞系NS1 （kappa(NS1)）表达的未组装的免疫球蛋白轻链在体内降解，其半衰期为50-60分钟，其降解方式与内质网（ER）相关降解非常相似（Knittler et al., 1995）。在这里，我们发现肽醛MG132和PS1以及特异性蛋白酶体抑制剂lactacystin有效地延长了kappa（NS1）的半衰期，证明了蛋白酶体介导的降解途径。亚细胞分离和蛋白酶保护实验表明，在蛋白酶体抑制下，kappa（NS1）在内质网定位。kappa（NS1）折叠状态分析和大小分级实验独立证实了这一点，结果表明，当蛋白酶体活性被阻断时，免疫球蛋白轻链仍与ER伴侣BiP结合。此外，在乳系统素处理的细胞中进行的动力学研究显示，BiP-kappa（NS1）复合物的物理稳定性随时间的增加而增加，这表明在较老的复合物中存在额外的蛋白质。总之，我们的数据支持一个内质网相关降解模型，其中可溶性非糖基化蛋白从BiP的释放及其在内质网外的反转录易位与蛋白酶体活性紧密耦合。
Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (Knittler et al., 1995). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1), upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.