Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone dependent ubiquitination by the action of E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70’s role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of SSE1, and the action of J proteins are all needed for Ubr1 mediated quality control. To discern if chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage, in which efficiently folded proteins are spared, while slow- or non-folding proteins are iteratively tagged with ubiquitin for subsequent degradation.

分子伴侣可以介导蛋白质折叠和降解。此过程称为蛋白质分类，需要进行研究以揭示用于基本科学和翻译目的的质量控制机制。在酵母中，许多错误折叠的蛋白质通过E3连接酶Ubr1和San1的作用进行伴侣依赖的泛素化，从而可以进行蛋白质分类的详细研究。在细胞中，HSP70和HSP90都介导了底物泛素化，并且规范的ATP循环对于HSP70的作用是必需的：我们发现，Hbr70的ATP水解，SSE1的核苷酸交换活性和J蛋白的作用都是Ubr1介导的。质量控制。为了确定伴侣分子是否直接参与了Ubr1介导的泛素化，我们开发了一种基于微珠的检测方法，该检测方法具有共价固定但可释放的错误折叠的蛋白质，以消除分子伴侣对底物物理状态或运输的可能影响。在这个在体外测定中，仅USP1介导的泛素化仅需要HSP70及其ATPase循环和相关的伴侣蛋白。遍在蛋白化过程中对HSP70 ATP循环的要求提示了一种可能的分类方法，其中可以保留有效折叠的蛋白，而慢折叠或非折叠蛋白则用遍在蛋白反复标记以进行后续降解。