Identification of regulatory kinases of the FoF1 ATP synthase beta subunit

• Autor(a) principal: Campos, Ana Isabel Oliveira
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/30855
• topic: S. cerevisiae; Mitochondria; ATP synthase β subunit; Phosphorylation; Phosphoregulation; Phos-Tag™; Regulatory kinases; Cdc5

In higher eukaryotes, approximately 90% of cellular ATP is produced by ATP synthase, a mitochondrial enzymatic complex. Numerous post-translational modifications, particularly phosphorylation, were reported for all ATP synthase subunits, both in physiological and pathological states. Still, the impact of these modifications for ATP synthase regulation or the signaling pathways involved remains ill-defined. Previously, two phosphorylation sites were identified in the catalytic β subunit of the FoF1 ATP synthase (Atp2) of S. cerevisiae, Threonine 124 (Thr) and Thr317. Phosphorylation of these residues resulted in Atp2p stabilization, increased ATP synthase activity and mitochondrial respiration. Yet, the kinases involved in the phosphorylation of these residues, conserved across species, are unknown. Using S. cerevisiae as a model, the aim of this study was thus to identify the kinase(s) involved in the phosphoregulation of the ATP synthase β subunit. Using bioinformatic motif prediction, four kinases were selected as potential Atp2 regulators: Cdc5, Ipl1, Hrr25 and Pkc1. Cdc28 was also evaluated since a genetic interaction with Atp2 was previously observed. Kinase expression was manipulated to evaluate possible effects in Atp2p phosphorylation and Atp2p levels. Zn2+Phos-Tag™ SDS-PAGE was optimized and used to detect Atp2p phosphorylation. The overexpression of Pkc1, Ipl1 and Cdc5 resulted in increased Atp2 phosphorylation. Overexpressing Ipl1 also caused an increase in Atp2p levels, consistent with increased phosphorylation, while a minor increase was observed for Cdc5. The role of Cdc5 on mitochondrial function was further evaluated. Overexpression of Cdc5 resulted in a significant increase in mitochondrial respiration and repressing Cdc5 prevented growth in respiratory media, indicating that Cdc5 plays an important, previously unsuspected role in mitochondrial function. Though overall evidence supports a role for Cdc5 in Atp2 regulation, further studies are required to confirm that Cdc5 is, in fact, a direct phosphoregulator of Atp2. Identification of Atp2p regulatory kinases will improve our understanding of the signalling pathways regulating ATP synthase function. Since many pathologies are associated with dysfunction of this vital complex, identification of its regulatory kinases can provide, in the long run, new therapeutic targets.