PROTEIN INTERACTION WITH NEWLY SYNTHESISED SUBUNITS OF THE OXIDATIVE PHOSPHORYLATION SYSTEM

Abstract

The cell's most efficient energy conversion occurs within the mitochondrion, through a process called oxidative phosphorylation (OXPHOS). OXPHOS involves five key components, complexes I-V, located within the inner mitochondrial membrane. The complexes consist of various subunits that are encoded by genes from both the nucleus and the mitochondria itself. Saccharomyces cerevisiae possesses seven, cytochrome b, Cox1-3, Atp 6, 8 and 9. In a recent unpublished study, co-translationally interactions with nascent mitochondria-encoded subunits in yeast was revealed. However, the other known assembly factors that interact with nascent subunits seem not to bind co-translationally with the nascent chains in this study. The question is whether other assembly factors are recruited post-translationally rather than cotranslationally. To address this question, an assay has been developed to detect proteins interacting with newly synthesized chains of mitochondrial-encoded OXPHOS subunits in a post-translational manner. The experimental approach involved isolating mitochondria from a strain of S. cerevisiae where cytochrome b was conjugated with hemagglutin (HA) epitope in the C-terminus for immunoprecipitation. Both photocrosslinking and chemical cross-linking methods were employed to determine the optimal approach. In photo-crosslinking, in organello translation was carried out to incorporate photo-reactive leucine analogue crosslinker and 35S-methionine into newly synthesised polypeptides of cytochrome b. For chemical crosslinking, a gene deletion was applied to restrict the assay to nascent chains and ten different cross-linkers were tested, with the analogues m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS) and succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) emerging as most suitable. Following crosslinking, the subunit and its interactors were isolated by immunoprecipitation using HA magnetic beads This was then planned to be subjected to mass spectrometry analysis, to identify the putative assembly factors that bind to nascent subunits post-translationally. This study investigates the methods of determining protein interactions between mitochondrial OXPHOS assembly factors and mitochondrial-encoded OXPHOS subunits and offers valuable insights into studying the molecular process of biogenesis of the OXPHOS system.