Determining the conserved function of mitochondrial LYR proteins

Abstract

Mitochondria are essential organelles found in many different eukaryotic organisms. They are the main site of ATP synthesis through oxidative phosphorylation (OXPHOS) involving a number of complexes (I-IV) on the mitochondrial inner membrane. The biogenesis of OXPHOS complexes is intricate process which utilises several assembly factors. LYRM proteins are small mitochondrial proteins which have been identified as either assembly factors or accessory domains of the respiratory chain subunits. According to the existing literature, there are about thirteen LYRM proteins have been found in humans, plants, and yeasts with diverse roles in OXPHOS complexes. The exact mechanism for these proteins has not been determined but several LYRM proteins have been shown to be involved in the assembly of many subunits across the respiratory chain complexes and the defects in LYRM proteins have been associated with many human diseases. Furthermore, there are several LYRM proteins in plants that do not have direct yeast or mammalian homologues. Therefore, the purpose of this research project was to determine if LYRM protein function is conserved in yeast (Saccharomyces cerevisiae) and plants (Arabidopsis thaliana). The objectives of this aim were to identify if various LYRM proteins can functionally complement their yeast counterparts and to test the ability of various LYRM proteins to interact with mitochondrial respiratory chain subunits as shown in yeast and mammalian systems. During this project, many experiments and assays were carried out including gene cloning, yeast complementation assays and yeast-2-hybrid interaction assays. The complementation assays showed that Arabidopsis SDHAF1 can functionally complement its yeast ortholog (ScSDHAF1) suggesting that the plant SDHAF1 may have similar roles in the Fe/S clusters insertion within Complex II subunits. Also, our results showed that the AtSDHAF3, At1g76065 and ScSDHAF1 can complement ScSDHAF3 function with some complementation were observed with other LYRM proteins, ScFMC1, AtISD11, AtSDHAF1, AtFMC1, AtMZM1, AtB14 and AtB22. This suggests that these LYRMs may have similar roles to ScSDHAF3. Moreover, the plant ISD11 was able to completely restore the isd11 deletion strain viability on glucose media. Last complementation assay of the mzm1 deletion strain showed that AtSDHAF1, AtISD11, ScFMC1 were able to rescue the defective growth phenotype. The yeast two hybrid assay results were not persuasive as many interactions were observed amongst many constructs suggesting these are not true interactions. All these results confirmed that the predicted Arabidopsis homologs to the yeast SDHAF1, SDHAF3, ISD11 and MZM1 can complement the growth defect observed in the yeast deletion strains. Furthermore, we identified that the unknown LYRM gene (At1g76065) may also have possible roles in the Fe/S clusters insertion as it was able to restore sdhaf1 and mzm1 deletion strains growth phenotype. Moreover, we identified that several other Arabidopsis LYRM could also partially complement the yeast phenotype growth deficiency of sdhaf3 and mzm1 deletion strains suggesting that LYRM proteins are functionally conserved among yeasts and plants. Overall, this research provides new information to the existing knowledge about the LYR proteins functions across yeasts and plants.