Mitochondrial inheritance and quality control

蛋白质稳态依赖于蛋白质合成、折叠和降解的精确控制。 蛋白质抑制错误导致蛋白质聚集，这是有毒的，并与神经退行性疾病，心血管疾病， 肌肉和代谢紊乱，以及过早衰老。内质网和线粒体是蛋白质的主要场所 折叠，并得到质量控制机制的支持，这些机制可以纠正蛋白质折叠或消除蛋白质， 受损无法修复的细胞器ER相关降解（ERAD）和线粒体相关降解 降解（MAD）是功能和机制相关的机制。在这两种情况下，错误折叠的蛋白质 鉴定、泛素化、从细胞器中提取并被蛋白酶体降解。然而，这两种途径 有局限性。以前的研究表明，MAD蛋白质稳态仅限于线粒体外膜， 膜（OM）蛋白，<10%的线粒体蛋白。此外，MAD和ERAD固有地是低吞吐量的 因为它们作用于单个蛋白质。我们的R35资助的研究表明，1）MAD在以下方面发挥着重要作用： 2）MAD功能的丧失导致过早衰老，和 3)MAD不仅在线粒体外膜中，而且在基质和细胞中起蛋白质抑制控制的作用。 细胞器的内膜。在补充研究中，我们发现了一个保守的ER蛋白抑制途径， （ER-PERM），其具有与ERAD重叠的功能，但具有更高的通量并有助于ER应激 酵母、哺乳动物细胞和细胞模型对新发现的先天性肌营养不良症的反应 (CHKB CMD）。在ER-PERM中，脂滴（LD），在ER膜上形成的细胞器，充当逃逸舱口 用于大规模去除未折叠的ER蛋白并降解这些蛋白及其LD载体。在这里， 降解通过微自噬发生，微自噬是一种保守但研究不足的自噬形式，不依赖于 自噬体或核心ATG基因，用于将货物递送至液泡（酵母溶酶体）。重要的未来 目的是1）了解线粒体内MAD功能的机制，2）生理学后果， 序列的MAD介导的线粒体蛋白酶抑制，和3）确定组件和功能的后果 关于ER-PERM我们要求资金更换一台损坏的、无法修复的、使用超过15年的酶标仪（Tecan NanoQuant），它被大量用于对完成我们的R-35资助的研究至关重要的测定 包括酵母生长曲线、蛋白质和核酸测定、酶测定和筛选 在感兴趣的蛋白质上表达荧光标签。当其他实验室或设备中有酶标仪时 核心，这些仪器不可用于长期，不间断的使用，如分析酵母生长曲线或 任何基于生长的筛查（350小时/月）。因此，我们要求资金购买新的读板器（Tecan Infinite Base Unit，M200和F200 PRO酶标仪）以及计算机硬件和软件来驱动 读板器。

Protein homeostasis, or proteostasis, relies on precise control of protein synthesis, folding and degradation. Proteostatic errors lead to protein aggregates, which are toxic and linked to neurodegenerative, cardiovascular, muscular and metabolic disorders, and to premature aging. The ER and mitochondria are major sites for protein folding and are supported by quality control mechanisms that correct protein folding or eliminate proteins or organelles that are damaged beyond repair. ER-associated degradation (ERAD) and mitochondria-associated degradation (MAD) are functionally and mechanistically related mechanisms. In both, misfolded proteins are identified, ubiquitinated, extracted from organelles and degraded by the proteasome. However, both pathways have limitations. Previous studies suggested that MAD proteostasis was restricted to mitochondrial outer mem- brane (OM) proteins, <10% of mitochondrial proteins. Moreover, MAD and ERAD are inherently low-throughput because they act on individual proteins. Our R35-funded research revealed that 1) MAD plays a major role in mitochondrial and cellular fitness in a model for aging, 2) loss of MAD function results in premature aging, and 3) MAD functions in proteostatic control not just in the mitochondrial outer membrane, but also in the matrix and inner membrane of the organelle. In complementary studies, we identified a conserved ER proteostasis pathway (ER-PERM) that has overlapping function with ERAD, but has higher throughput and contributes to the ER stress response in yeast, mammalian cells and cellu models for a newly identified congenital muscular dystrophy (CHKB CMD). In ER-PERM, lipid droplets (LDs), organelles that form at ER membranes, act as escape hatches for large-scale removal of unfolded ER proteins and degradation of those proteins and their LD carriers. Here, degradation occurs by microautophagy, a conserved but understudied form of autophagy that does not rely on autophagosomes or core ATG genes for delivery of cargoes to the vacuole (yeast lysosome). Important future goals are to 1) understand the mechanism of MAD function within mitochondria, 2) the physiological conse- quences of MAD-mediated mitochondrial proteostasis, and 3) identify components and functional consequences of ER-PERM. We request funds to replace a broken, unrepairable, >15 year-old microplate reader (Tecan NanoQuant) that was heavily used for assays that are essential to the completion of our R-35-funded research including yeast growth curves, protein and nucleic acid determination, enzyme assays, and screening for expression of fluorescence tags on proteins of interest. While there are plate readers in other labs or equipment cores, those instruments are not available for long-term, uninterrupted use like analysis of yeast growth curve or any growth-based screens (350 hrs/month). Thus, we request funds to purchase a new plate reader (Tecan Infinite Base Unit, M200 and F200 PRO Microplate Reader) and the computer hardware and software to drive the plate reader.