The Energetic Cost of Protein Retrotranslocation during ER-associated Degradation

描述（由申请人提供）：大约三分之一的新合成蛋白质通过分泌途径。这些蛋白质在其初级结构以及保守的伴侣系统中使用线索，以便折叠成其天然的功能构象。当折叠被破坏时，即由于遗传基因突变，错误折叠的蛋白质被细胞蛋白质平衡机制“感知”，然后可能成为er相关降解（ERAD）的目标。ERAD底物根据其内质网管、膜或细胞质结构域内折叠病变的位置进行分类。具体来说，ERAD底物似乎可以在合成过程中的不同点被识别，这取决于“折叠损伤”的位置。Ste6p是在酿酒酵母中发现的一种ABC转运蛋白，是a因子交配信息素输出所必需的。截断Ste6p的大c端胞质结构域（Ste6p*）可将该蛋白转化为用于根除- c（细胞质）途径的底物。由于将Ste6p*转化为ERAD底物的突变位于末端c端，因此降解的“决定”必须发生在翻译后。最近来自Brodsky实验室的证据表明，当Ste6p*的截断的c端被转移到其他蛋白质时，它足以诱导嵌合体的降解，这提高了该序列可以作为降解目标蛋白质的“degron”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。本研究的第一个目标是确定具有翻译后ERAD- c型病变的ERAD底物的伴侣、泛素化机制和逆转录易位的要求。其次，由于对整体膜疏水性如何影响ERAD效率知之甚少，我建议生成包括具有不同跨膜结构域的Ste6p* degron在内的嵌合结构体。这些研究的目的是确定ERAD底物的跨膜疏水性差异是否与降解和/或膜提取效率相关。这些研究将有助于扩展我们对细胞降解错误折叠蛋白的复杂机制的认识，确定催化ERAD的新因子，并确定疾病相关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.