UCLA

Coenzyme Q (CoQn or ubiquinone) is an essential lipid molecule containing a redox-active benzoquinone head group and polyisoprenoid tail of varying length, denoted by n. The structural features of CoQ afford its ability to perform its role as a mobile electron carrier in the mitochondrial electron transport chain. Its synthesis is driven by the nuclear-encoded Coq polypeptides, several of which are required to assemble into a protein-lipid complex, the CoQ synthome. The formation of the CoQ synthome is coordinated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a membrane contact site that bridges the ER and mitochondria. CoQ deficiency results in various clinical manifestations, therefore it is imperative to have a holistic understanding of how CoQ is endogenously synthesized and distributed, and how these processes are regulated. We utilize Saccharomyces cerevisiae as a model organism given the high functional conservation of COQ genes and mitochondrial membrane contact sites with humans. Chapter 1 details an overview of the members required for CoQ biosynthesis and discusses the relationship between the CoQ synthome and ERMES. Chapter 2 focuses on the recharacterization of Coq10 using nonfunctional Coq10 isoforms. The COQ10 gene is positioned adjacent to MDM12, which encodes the cytosolic subunit of ERMES. Due to this arrangement, we discovered that deletion of COQ10 impacts MDM12 expression, resulting in diminished Mdm12 protein content and ERMES dysfunction. We use CRISPR-Cas9 to introduce mutations within COQ10 that preserve MDM12 expression, thereby maintaining ERMES. We show that Coq10 is not required for CoQ synthome assembly, and phenotypes associated with the coq10Δ mutant were due to the impact on ERMES. Considering ERMES may have a specific role in CoQ synthome assembly, Chapter 3 evaluates if ERMESΔ mutants can be rescued by deleting COQ11. The COQ11 gene product is proposed to be a negative modulator of CoQ synthome assembly, indicated by the ability of the COQ11 deletion to rescue complex stability in the coq10Δcoq11Δ double mutant. With Dr. Maya Schuldiner, we show that select ERMESΔ mutants can be rescued by deleting COQ11, indicating the role of ERMES in regulating CoQ synthome assembly can be circumvented when ERMES is absent. Chapter 4 examines how the expression of an artificial tether affects CoQ biosynthesis. Artificial tethers have been used to study membrane contact sites in vivo, and we report that the act of physical tethering does not bolster CoQ biosynthesis, and instead may impact trafficking and degradation. The results from Chapter 4 support the notion that ERMES has a direct role in CoQ synthesis and distribution, which an artificial tether cannot fulfill. Appendices I-III contain previous publications, including: characterization of the coq10Δcoq11Δ phenotype, an enzymology study from my undergraduate research published during my graduate program, and a structural analysis of clinically relevant single nucleotide variants in human COQ genes. Together, this work helps to clarify the roles of Coq10 and ERMES in the regulation of CoQ biosynthesis and distribution.