Understanding SNX-BAR protein structure and function in yeast

自噬是在细胞日常维护或饥饿等环境压力下从细胞中去除老化或受损成分的细胞过程。自噬的循环副产品也可以用来维持细胞生存的关键代谢。自噬从酵母到人类都是保守的，必须严格控制，以防止可能导致异常细胞生长或死亡的生物错误。分选连接蛋白是一个与自噬许多方面直接相关的蛋白家族，但其作用机制尚不清楚。本项目旨在通过确定原子结构和分析新发现的分类连接蛋白的功能来确定自噬过程中控制分类连接蛋白功能的调控元件。该项目还将为研究生和本科生提供生物物理和生化研究方面的培训机会。该项目将通过开发新颖的3D打印和虚拟现实（VR）探究式学习模块来改善本科课程，从而对实验室以外的领域产生影响。此外，该项目将创建一个以大分子结构和功能为中心的基于课程的本科体验（CURE）。总的来说，更广泛的影响将使在职科学家，包括那些来自STEM中代表性不足的背景的科学家，有机会加强他们的科学教育。分类连接蛋白（SNXs）构成了一个不同的分子家族，在膜运输、细胞信号传导、膜重塑和细胞器运动中发挥着不同的作用。分类蛋白的SNX-BAR亚家族通常被认为在内体分类中起作用，但最近有几个亚家族被证明参与自噬体的生物发生。然而，它们在自噬中的作用机制尚不清楚。现有的模型表明，进化上保守的Phox-Homology （PX）结构域是所有分选连接蛋白的共同特征，它特异性地与内体的主要脂质部分磷脂酰肌醇3-磷酸（PI3P）结合，从而驱动SNX-BAR功能。本研究的目的是了解一种名为Vps501的酵母SNX-BAR蛋白的结构和功能，以及它与参与自噬的SEA复合物的相互作用。初步数据表明，Vps501具有独特的结合脂质的特异性，需要改变构象才能与液泡膜发生独特的相互作用。本项目将结合生物化学、遗传和结构生物学实验来实现其目标。该项目将连接SNX-BAR蛋白的脂质特异性模式和结构特征，同时为理解空泡内系统与自噬之间的关系提供新的范例。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Autophagy is the cellular process of removing aged or damaged components from the cell during routine cellular maintenance or during times of environmental stress such as starvation. The recycled by-products of autophagy can also be used to sustain critical metabolism for cell survival. Autophagy is conserved from yeast to humans and must be tightly controlled to prevent biological miscues that can result in aberrant cellular growth or death. The sorting nexins are a family of proteins that have been directly implicated in many aspects of autophagy, however the mechanism of their action there is poorly understood. This Project seeks to determine the regulatory elements that govern sorting nexin function during autophagy by determining the atomic structure and analyzing the function of a newly discovered sorting nexin. The Project will also provide graduate and undergraduate students an opportunity for training in biophysical and biochemical research. The Project will have an impact outside the laboratory by improving undergraduate curriculum through the development of novel 3D printing and Virtual Reality (VR) inquiry-based learning modules. In addition, the Project will create a course based undergraduate experience (CURE) centered on macromolecular structure and function. Overall, the broader impact will give scientists-in-training, including those from backgrounds underrepresented in STEM, an opportunity to enhance their scientific education.The sorting nexins (SNXs) constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling and organelle motility. The SNX-BAR subfamily of the sorting proteins are typically thought to function in endosome sorting, but recently several have been shown to participate in autophagosome biogenesis. However, the mechanism of their role in autophagy is unclear. Prevailing models indicate that an evolutionarily conserved Phox-Homology (PX) domain, a feature common in all sorting nexins, binds specifically to phosphatidylinositol 3- phosphate (PI3P), the major lipid moiety of the endosome, to drive SNX-BAR function. The goal of this research is to understand the structure and function of a previously uncharacterized yeast SNX-BAR protein termed Vps501 and its interaction with the SEA complex that is involved in autophagy. Preliminary data suggests that Vps501 demonstrates a unique specificity for binding lipids and requires conformational changes to uniquely interact with the vacuole membrane. This Project will use a combination of biochemical, genetic and structural biology experiments to accomplish its goal. The Project will connect patterns of lipid specificity and structural characteristics in SNX-BAR proteins, while providing a new paradigm for understanding the relationship between the endo-vacuolar system and autophagy.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.