The Energetic Cost of Protein Retrotranslocation during ER-associated Degradation

描述(申请人提供)：大约三分之一的新合成的蛋白质通过分泌途径。这些蛋白质利用其一级结构中的线索以及保守的伴侣系统来折叠成它们天然的、功能的构象。当折叠被破坏时，即由于遗传的基因突变，错误折叠的蛋白质被细胞蛋白平衡机制“感知”，然后可能成为内质网相关降解(ERAD)的靶标。ERAD底物已经根据折叠病变在其ER管腔、膜或细胞质区域中的位置进行了分类。具体地说，似乎ERAD底物在合成过程中可以在不同的位置被识别，这取决于“折叠病变”的位置。Ste6p是在酿酒酵母中发现的一种ABC转运蛋白，是a因子交配信息素出口所必需的。截断Ste6p的大C末端细胞质结构域(Ste6p*)将蛋白质转化为ERAD-C(细胞质)途径的底物。由于将Ste6p*转化为ERAD底物的突变位于极端的C末端，因此降解的“决定”必须发生在翻译后。来自Brodsky实验室的最新证据表明，当Ste6p*的截短C末端转移到其他蛋白质上时，足以诱导嵌合体的降解，从而增加了该序列可以作为目标蛋白质降解的可能性。我建议剖析含有Ste6p*degron的嵌合蛋白所采取的ERAD途径。这项研究的第一个目标是确定具有翻译后ERAD-C型损伤的ERAD底物的伴侣、泛素化机制和逆转移位的要求。其次，由于人们对膜的疏水性如何影响ERAD效率知之甚少，我建议构建包括Ste6p*degron在内的具有不同跨膜域的嵌合结构。这些研究的目的是确定ERAD底物跨膜疏水性的差异是否与降解和/或膜提取效率相关。这些研究将有助于扩大我们对细胞用来降解错误折叠蛋白质的复杂机制的知识，识别催化ERAD的新因素，并确定与疾病相关的ERAD底物的潜在药物靶点。

DESCRIPTION (provided by applicant): Approximately one-third of all newly synthesized proteins pass through the secretory pathway. These proteins use cues within their primary structure as well as a conserved chaperone system in order to fold into their native, functional conformations. When folding is disrupted, i.e. by an inherited genetic mutation, misfolded proteins are "sensed" by the cellular proteostasis machinery and may then be targeted for ER-associated degradation (ERAD). ERAD substrates have been classified based on the location of the folding lesion within their ER lumenal, membrane, or cytoplasmic domains. Specifically, it appears that an ERAD substrate can be recognized at different points during its synthesis depending on the location of the "folding lesion". Ste6p is an ABC transporter found in the yeast S. cerevisiae, and is required for export of the a-factor mating pheromone. Truncation of Ste6p's large C-terminal cytoplasmic domain (Ste6p*) converts the protein into a substrate for the ERAD-C (cytoplasmic) pathway. Because the mutation that renders Ste6p* into an ERAD substrate resides at the extreme C-terminus, the "decision" for degradation must occur post-translationally. Recent evidence from the Brodsky lab indicates that when the truncated C-terminus of Ste6p* is transferred to other proteins it is sufficient to induce the degradation of the chimera, raising the possibility that the sequence can act as a "degron" to target proteins for degradation. I propose to dissect the ERAD pathway taken by chimeric proteins containing the Ste6p* degron. The first goal of this research is to determine the chaperones, ubiquitination machinery, and requirements for retrotranslocation for an ERAD substrate with a post-translational ERAD-C-type lesion. Second, since little is known about how integral membrane hydrophobicity influences ERAD efficiency, I propose to generate chimeric constructs including the Ste6p* degron with varied transmembrane domains. The goal of these studies is to determine if the difference in transmembrane hydrophobicity of an ERAD substrate correlates with degradation and/or membrane extraction efficiency. These studies will help to extend our knowledge of the complex mechanisms used by cells to degrade misfolded proteins, identify new factors that catalyze ERAD, and identify potential drug targets for disease-associated ERAD substrates.