Mitochondrial inheritance and quality control

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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.