Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1–coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6. The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because “fused” proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.
Summary
Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity.
For complete details on the use and execution of this protocol, please refer to
Divakaruni et al. (2018)
.
Coenzyme Q, also known as ubiquinone or Q, is a redox‐active lipid component of the electron transport chain that functions in cellular energy metabolism. Due to its redox capabilities, reduced Q (or QH2) also serves as an important lipid‐soluble antioxidant effective in alleviating lipid peroxidative damage. In Saccharomyces cerevisiae, thirteen known mitochondrial proteins (Coq1–Coq11, Yah1, and Arh1) drive Q biosynthesis. Many of these Coq polypeptides required for Q production are localized to the matrix side of the mitochondrial inner membrane where they form a high molecular weight, multi‐subunit complex known as the ‘CoQ‐synthome’. Absence of individual Coq proteins causes severe defects in Q biosynthesis, and such coq null mutants lose the ability to respire. However, the functional roles of some Coq polypeptides as well as several steps in the Q biosynthetic pathway remain unknown. Here, we present the identification of a novel mitochondrial protein that associates with the CoQ‐synthome encoded by the open reading frame YLR290C, which we renamed Coq11. Co‐immunoprecipitation and lipid analyses demonstrate that Coq11 interacts with other Coq polypeptides as well as Q and Q‐intermediates, and is required for efficient de novo Q biosynthesis in the BY4742 genetic background. We have begun work towards understanding the function of Coq11 in CoQ‐synthome formation, as well as the role of Coq11 as a putative redundant decarboxylase in Q biosynthesis. Taken together, this project will provide powerful insights into Q biosynthesis and regulation towards the objective of designing effective therapeutics for diseases caused by Q deficiencies.Support or Funding InformationThis research was supported by NSF MCB‐1330803 and the Ruth L. Kirschstein National Research Service Award GM007185.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.