Mode of targeting to the proteasome determines GFP fate
Mode of targeting to the proteasome determines GFP fate
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DOI：
10.1074/jbc.ra120.015235
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发表时间：
2020-11-20
影响因子：
4.8
通讯作者：
Kraut, Daniel Adam
中科院分区：
文献类型：
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作者：
Braganca, Christopher Eric;Kraut, Daniel Adam
泛素-蛋白酶体系统是真核细胞中蛋白质降解的典型途径。在蛋白酶体降解试验中，绿色荧光蛋白经常被用作报告蛋白。然而，目前使用的GFP有多种变体，这些变体具有不同的内在稳定性。此外，有多种方法可以将底物靶向于蛋白酶体，这些差异也可能影响蛋白酶体展开和降解底物的能力。在这里，我们研究了不同内在稳定性的GFP变体的命运是如何由靶向蛋白酶体的模式决定的。我们比较了两种靶向系统：线性Ub(4) degrons和酵母Rad23的UBL结构域，这两种系统都是降解实验中常用的。令人惊讶的是，UBL降解体可以降解最稳定的含有sgfp的底物，而Ub(4)降解体则不能。通过循环排列破坏GFP的稳定性，使得任一靶向信号都可以降解，这表明结构域稳定性和靶向模式共同决定了底物的命运。难以展开的底物被多次释放并重新接合，降解起始区域的去除提供了一种替代的剪切途径，以防止展开和降解；UBL降解有利于降解甚至难以展开的底物，而Ub(4)降解则有利于剪切。最后，我们发现泛素受体Rpn13主要负责增强蛋白酶体降解稳定的ubl标记底物的能力。我们的研究结果表明，靶向方法和报告蛋白的选择对蛋白质降解实验的设计至关重要。
The ubiquitin-proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub(4) degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub(4) degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub(4) degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.