Increasing Mitochondrial Substrate-level Phosphorylation Can Rescue Respiratory Growth of an ATP Synthase-deficient Yeast

Abstract

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F 0 F 1 -ATP synthase at elevated temperatures. The ⌬fmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37°C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the ⌬fmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports ␣-ketoglutarate and ␣-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the ␣-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.In the inner mitochondrial membrane, the F 0 F 1 -ATP synthase performs the late step of the oxidative phosphorylations. This hetero-oligomer uses the electrochemical transmembrane proton gradient generated by the respiratory chain to catalyze ATP synthesis from ADP and inorganic phosphate (for reviews, see Refs. 1 and 2). It is composed of two distinct domains, a membrane-integrated F 0 domain containing a proton channel and a hydrophilic peripheral F 1 domain bearing the catalytic sites for ATP synthesis.This enzyme comprises about 20 different subunits for an overall molecular mass approaching 600 kDa. The genes encoding these proteins are located part in the nucleus and part in the mitochondrion itself. The subunits of nuclear origin are synthesized in the cytoplasm and then imported into the mitochondrion (for review, see Ref.3), whereas the mitochondrial DNA-encoded subunits are synthesized within the mitochondrion. This genetic compartmentalization makes the assembly of the ATP synthase complex a particularly intricate process. Studies in Saccharomyces cerevisiae have shown that specific proteins, usually called assembly factors, not belonging to the final complex, are required in the different steps of the enzyme biogenesis. Three such proteins, Atp11p, Atp12p, and Fmc1p, were shown to facilitate the assembly of the ATP synthase F 1 component (4 -6), which is made of five different nuclear-encoded proteins in the ␣ 3 ␤ 3 ␥␦⑀ stoichiometry (7). The ␣ and ␤ subunits alternate in ...

The mitochondrial phosphate carrier: Role in oxidative metabolism, calcium handling and mitochondrial disease

“…This could prevent a large depolarization with consequent mitochondrial ATP consumption [58]; such a mechanism would spare cytosolic ATP pools. Interestingly, in yeast, destabilization of the ATP synthase was compensated by ATP production via substrate level phosphorylation of ADP within mitochondria [70], demonstrating that this type of metabolic reconfiguration can arise in an intact organism.…”

Cancer as a Metabolic Disease: Implications for Novel Therapeutics

“…It is now recognized from numerous studies that oxygen consumption rates are not always linked to a normally coupled oxidative phosphorylation (83)(84)(85)(86). It can be difficult to determine the degree to which mitochondrial ATP production arises from coupled respiration or from TCA cycle substrate level phosphorylation (87)(88)(89)(90). The origin of mitochondrial ATP production in tumor cells requires further clarification in light of these issues.…”

“…It would therefore be important for investigators to exclude the influence of a Crabtree effect on the assessment of energy measurements in cultured cells. Although a Crabtree effect might suppress OxPhos, the TCA cycle should remain functional and produce ATP through substrate level phosphorylation (87)(88)(89)(90). Under certain conditions (hypoxia), the tumor TCA cycle can work in both forward and reverse (reductive) directions (129,130).…”

Cancer as a Metabolic Disease

“…In addition to its role in replenishing TCA cycle intermediates (anaplerosis), glutamine can also provide energy through stimulation of glycolysis in the cytoplasm and through substrate level phosphorylation in the TCA cycle (glutaminolysis) [45][46][47][48][49]. Energy obtained through substrate level phosphorylation in the TCA cycle can compensate for deficiencies in either glycolysis or oxidative phosphorylation [46,48,50], and can represent a major source of energy for the glutamine-dependent cancers.…”