Investigation of Mitochondrial Ribosome Regulators at the Inner Membrane

Mitochondrial biogenesis relies on the proper assembly of proteins from dual genetic origin. Nuclear-encoded proteins are imported into the organelle and incorporated into multiprotein complexes together with proteins synthesized on mitochondrial ribosomes. The OXPHOS machinery is composed of such multiprotein complexes (I-V) which are embedded into the inner mitochondrial membrane. To guarantee the functionality of the OXPHOS system, the assembly of these protein complexes is a highly coordinated process and dependent on the attendance of so-called respiratory chain assembly factors. Over the years, an enormous amount of research has been devoted to investigating the detailed mechanisms of individual assembly steps of each OXPHOS complex and by this, many assembly factors were identified. Because most of the research has been gleaned from studies using the model organisms S. cerevisiae, it became fascinating to look into higher eukaryotes as well. In this study, the interactome analysis of the human mitochondrial large ribosomal subunit mL62 identified TMEM223 as a novel potential assembly factor for complex IV and SMIM4, a protein of small molecular weight, involved in complex III biogenesis. By defining the interactors of SMIM4FLAG we further explored C12ORF73, which was previously described as essential complex III assembly factor in vertebrates (Zhang et al., 2020). Our main interest laid in the characterization of the biochemical and mechanistic role of these proteins regarding the respective complex assembly. For this purpose, transient siRNA approaches and a TMEM223 knockout cell line (TMEM223- /- ) were used to analyze apparent changes in mitochondrial biogenesis. Furthermore, the potential timeframes when these assembly factors are acting were defined by analyzing the interactome of SMIM4FLAG and C12ORF73FLAG. One of the crucial steps in complex biogenesis is the insertion of mitochondrial-encoded proteins into the inner mitochondrial membrane. From studies in S. cerevisiae it is assumed that the human homolog OXA1L plays a leading role in this process. Mass spectrometry analyses provided insights into the interactome of the OXA1L- complex and identified TMEM126A as a potential interaction partner. This study explores the versatile function of TMEM126A concerning the OXA1L interaction linked to the biogenesis of the OXPHOS machinery and mitochondrial translation.2022-09-1