A mitochondrial CO₂‐adenylyl cyclase‐cAMP signalosome controls yeast normoxic cytochromecoxidase activity

Abstract

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

Mitochondrial ribosome assembly in health and disease

“…Recent comprehensive affinity purification studies of GRSF1 and DDX28 followed by mass spectrometry analysis by two independent groups have yielded similar results [127,128]. In the study performed by our group, DDX28 was found associated with four major groups of relevant proteins [165]. The first group included most mitoribosomal proteins (45 mtLSU and 29 mtSSU MRPs).…”

“…Affinity purification of tagged GRSF1 identified ribosomal proteins [137] and the 16S rRNA methyltransferases MRM1, MRM2 and MRM3/RMTL1 [164]. Affinity purification of tagged MRM3 co-eluted ribosomal proteins and ribosome assembly factors that colocalize to the RNA granules, such as GRSF-1 and the DEAD-box helicase DDX28 [164,165]. Recent comprehensive affinity purification studies of GRSF1 and DDX28 followed by mass spectrometry analysis by two independent groups have yielded similar results [127,128].…”

“…However, it is more likely that RNA granules cosedimented with the nucleoids in sucrose gradients and therefore the SILAC results cannot exactly discriminate where mitoribosome assembly occurs. Studies of the composition of human DDX28 [165] and GRSF interactomes [128] mentioned earlier point toward the conclusion that most steps of mitoribosome biogenesis could actually occur in the RNA granules. It is necessary, however, to stress the concept of RNA granules as dynamic structures.…”

The subunit composition and function of mammalian cytochrome c oxidase

“…cAMP-dependent regulation of COX is even more complex and was shown to affect COX on the matrix side through a soluble adenylyl cyclase, which itself is regulated by the intramitochondrial CO 2 concentration (Acin-Perez et al, 2009), linking Krebs cycle activity to electron transport chain function. This mechanism is also present in yeast where two phosphorylation sites, Thr65 and Ser43, were identified on subunit IV-1 (yeast nomenclature: COX subunit Va) (Hess et al, 2014).…”

Mitochondrial cAMP signaling

“…It involves a mitochondrially localized sAC that can be directly activated by HCO 3 − [12] and was shown to synthesize cAMP locally in response to the CO 2 /HCO 3 − produced in the TCA cycle in mammalian cultured cells [95–100]. The matrix CO 2 -sAC-cAMP-PKA cascade is also involved in the allosteric regulation of COX activity by ATP in the S. cerevisiae, demonstrating a conserved role of cAMP signaling in fine-tuning energy metabolism [101]. However, the Drosophila and C. elegans genomes do not encode any known sAC [102], and the prevalence of intra-mitochondrial cAMP signaling in metazoans has been called into question.…”