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 Ub4 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 Ub4 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 Ub4 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.

中文翻译：

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靶向蛋白酶体的模式决定了 GFP 的命运。

泛素-蛋白酶体系统是真核细胞中蛋白质降解的典型途径。GFP 经常用作蛋白酶体降解试验中的报告基因。然而，有多种 GFP 变体在使用，这些变体具有不同的内在稳定性。此外，底物靶向蛋白酶体的方式有多种，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 Ub4 degron 和来自酵母 Rad23 的 UBL 域，这两种系统都常用于降解实验。出奇，UBL degron 允许降解最稳定的含有 sGFP 的底物，而 Ub4 degron 则不能。通过循环置换使 GFP 不稳定，允许降解与任一靶向信号，表明结构域稳定性和靶向模式相结合，以确定底物命运。难以展开的底物被多次释放和重新接合，去除降解起始区域提供了一种替代的剪切途径，可防止展开和降解；UBL degron 有利于甚至难以展开的底物降解，而 Ub4 degron 有利于剪切。最后，我们表明泛素受体 Rpn13 主要负责增强蛋白酶体降解稳定的 UBL 标记底物的能力。