Mitochondrial energy metabolism is crucial for providing the cell with the fuel it needs to perform a host of biological processes, and defects in this pathway can have serious consequences for many disease states, including neurodegeneration and diabetes. Therefore, understanding the roles of the components involved in energy generation is highly valuable for designing interventions in these disorders. Recent research has yielded new discoveries in the entry of pyruvate into the mitochondrial matrix as well as the synthesis and membrane placement of OXPHOS subunits, deepening our understanding of how mitochondrial energy metabolism proceeds and providing new potential targets for treatment of mitochondria-related diseases.
Adenosine triphosphate (ATP), whose hydrolysis fuels many cellular processes, is produced by a few key metabolic pathways in the cell. Aerobic respiration comprises glycolysis, which takes place in the cytosol, and the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), which occur in the mitochondria. Glycolysis and the TCA cycle, also known as the citric acid cycle or the Krebs cycle, each produce 2 molecules of ATP along with products important for further processes; glycolysis yields pyruvate, which is transported to the mitochondrial matrix and converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA feeds into the TCA cycle, which yields NADH as well as FADH2 for the electron transport chain (ETC). The ETC generates a proton gradient across the membrane, which drives conversion of ADP to ATP by the ATP synthase, producing 30+ ATP molecules (1). ATP is then used to drive biosynthesis, cell division, and many other processes in the cell.
Understanding the factors involved in development and function of mitochondrial energy production pathways is increasingly important due to the many diseases associated with defects in this machinery. Neurodegenerative diseases are especially closely linked with mitochondrial dysfunction. OXPHOS mutations are implicated as susceptibility factors in Alzheimer's and Parkinson's disease, and are linked to others like Leber hereditary optic neuropathy (LHON) and NARP syndrome (1). In Huntington disease, energy metabolism in the mitochondria is thought to be impaired at several different stages, including general downregulation of mitochondrial gene expression by inhibition of PGC-1a, diminished levels of complexes II and III of the electron transport chain, or inhibition of the TCA cycle by oxidative stress (2). Defects in mitochondrial energy metabolism are also associated with type II diabetes (3), and a recent study demonstrated that a certain subset of diffuse large B-cell lymphoma showed higher protein levels of respiratory chain and TCA cycle components, and derived a greater proportion of ATP from mitochondrial oxidative metabolism. Moreover, this subset was sensitive to disruption of fatty acid oxidation (4), suggesting the usefulness of targeting mitochondrial energy metabolism components in some types of cancer. Recently, new discoveries have elucidated two key steps in the energy production process: the transport of pyruvate into the mitochondrial matrix, and the generation of OXPHOS subunits for the electron transport chain.
Back to top Identification of the mitochondrial pyruvate carrier
After production of pyruvate by the cytosolic glycolysis pathway, which also generates a small amount of ATP, pyruvate is transported into the mitochondrial matrix and converted into acetyl-CoA by the pyruvate dehydrogenase complex (PDH). Acetyl-CoA contributes to the tricarboxylic acid (TCA) cycle, which generates NADH+ and FADH2 for oxidative phosphorylation. However, until recently, the proteins responsible for pyruvate transport into mitochondria had remained unclear. Two publications, one by Herzig and colleagues (5) and the other from Bricker et al. (6), identified the proteins, MPC1, MPC2, and MPC3, in a range of eukaryotic organisms from yeast to human.
Herzig et al. found that Mpc1, previously called Brp44L, was present in the inner mitochondrial membrane (IMM). Yeast strains lacking MPC proteins had impaired growth on synthetic dextrose media (SD), suggesting a role for MPCs in carbohydrate metabolism, and also displayed defects in lipoic acid production. The team tested whether MPC was involved in pyruvate transport into the matrix, and indeed, deleting these proteins hindered mitochondrial pyruvate uptake. To definitively show that the MPC proteins were the mitochondrial pyruvate carrier, the team expressed mouse MPC1 and 2 singly or together in Lactococcus lactis. Not only did coexpression of the proteins allow the bacteria to take up pyruvate, but the uptake displayed the same characteristics previously observed in the mitochondrial pyruvate carrier: it could be disrupted by UK-5099 and its activity was dependent on pH and the proton gradient.
In a simultaneous study, Bricker et al. also showed IMM localization of yeast Mpc1, as well as IMM localization of Mpc2 and interaction between these proteins, suggesting that they form a complex. Similarly to Herzig et al.'s study, they found that these genes were important for growth on a glucose or leucine-free medium. The ortholog in fruit flies, dMPC1, also appeared to be crucial for carbohydrate metabolism - mutant flies died on a sugar diet, and showed abnormally high levels of carbohydrates and pyruvate with lowered levels of ATP and TCA cycle intermediates. High pyruvate levels were observed in yeast along with reduced acetyl-CoA, yet normal PDH levels. As in Herzig's study, Bricker et al. found that MPC1 was needed for mitochondrial pyruvate uptake, and was sensitive to the MPC inhibitor UK-5099.
Finally, confirming the relevance of these findings in humans, the authors examined 4 children from 3 different families, each with a mitochondrial pyruvate oxidation deficiency. They found mutations in Brp44L/MPC1 in the affected individuals, and that expression of wild-type MPC1 in the cells of three of the subjects improved or restored pyruvate oxidation (6). Between these two studies, the MPC proteins are established as the mitochondrial pyruvate carrier, with implications for future studies into therapy for mitochondrial diseases.
Back to top CRIF1 and OXPHOS polypeptides - a critical link
Oxidative phosphoryation is the most efficient step for ATP production during aerobic respiration, and defects in the OXPHOS complexes that participate in this chain have been linked to a number of degenerative diseases (1). A new paper by Kim et al. describes the key role of mitoribosome-associated protein CRIF1 in the production of OXPHOS polypeptides, and demonstrates its importance in generation of a functioning oxidative phosphorylation chain (7).
After demonstrating CRIF1's association with the inner mitochondrial membrane, the team determined its function using brain-specific Crif1 knockout mice. Strikingly, a lack of CRIF1 in the brain led to significant neurodegeneration in the hippocampus and cerebral cortex, accompanied by diminished levels of OXPHOS subunits and complexes. Accordingly, Crif1-deficient mouse embryonic fibroblasts had lower oxygen consumption rates compared to wild-types as well as lower ATP levels in glucose-free media, the latter of which could be rescued by glycolysis in glucose-containing media.
Consistent with these results, Crif1-deficient cells showed decreases in OXPHOS complex activity, overall protein level, and assembly. Because mRNA levels were normal, however, the team determined that CRIF1 was likely involved in promoting proper OXPHOS function at a step following transcription. Indeed, they found a reduction in translation products via organelle translation assays with isolated mitochondria. Moreover, when they labeled translation products and separated out soluble from membrane-embedded polypeptides, they found that these products were in the membrane in wild-type cells, but in the soluble fraction in Crif1-deficient cells. The soluble fraction polypeptides were identified as OXPHOS subunits.
Immunoprecipitation showed multiple CRIF1 interactions, including proteins surrounding the mitoribosome polypeptide exit tunnel (like MRPL23, L28, and L44), the OXPHOS polypeptides themselves, and the mitochondrial chaperone Tid1, supporting a role for CRIF1 in helping insert OXPHOS polypeptides into the inner membrane after synthesis.
With these discoveries, the overall picture of how energy production proceeds in the mitochondrion at two distinct stages has become clearer. The identification of the MPC proteins, as well as the characterization of CRIF1's importance in developing a functioning set of OXPHOS protein complexes, yields new potential targets for diseases affected by mitochondrial dysfunction, and future studies will be needed to determine if and how these proteins intersect with a variety of mitochondria-related disorders.
- Koopman, W.J.H., Distemaier, F., Smeitink, J.A.M., and Willems, P.H.G.M. (2012) OXPHOS mutations and neurodegeneration. EMBO J. published online.
- Mochel, F. and Haller, R.G. (2011) Energy deficit in Huntington disease: why it matters. J. Clin. Invest. 121, 493.
- Szendroedi, J., Phielix, E., and Roden, M. (2011) The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 92.
- Caro, P. et al. (2012) Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma. Cancer cell 22, 547.
- Herzig, S. et al. (2012) Identification and function of the mitochondrial pyruvate carrier. Science 337, 93.
- Bricker, D.K. et al. (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, drosophila, and humans. Science 337, 96.
- Kim, S.J. et al. (2012) CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides into the mammalian mitochondrial membrane. Cell Metab. 16, 274.