Functional genomics of gene families encoding mitochondrial proteins / Dina Elhafez

2010

[Truncated abstract] Mitochondria are organelles present in almost all eukaryotic cells. In plants it is estimated that there are 1500 to 2000 different mitochondrial proteins. The majority of these proteins are encoded by nuclear-located genes, transcribed in the nucleus, translated in the cytosol and imported into mitochondria by the combined actions of several multi-subunit protein complexes, termed translocases. In plants, many mitochondrial proteins are encoded by gene families. In some cases, these families are large, such as those encoding pentatricopeptide repeat (PPR) proteins, and the function of each protein is proposed to be involved in different processes relating to transcription and translation in mitochondria. In other cases, the gene families are small ranging from 2 to several members and it is unclear if the proteins encoded by different genes have different functions. The overall aim of the research carried out in this thesis was to carry out investigations to determine if the proteins encoded by 2 small gene families in Arabidopsis thaliana (Arabidopsis) have different functions. Two gene families were selected for study, a gene family encoding alternative or type II NAD(P)H dehydrogenases (NDs) and the gene family encoding members of the preprotein and amino acids transporters (PRAT). Seven genes encode ND type proteins in the Arabidopsis genome. Six full length cDNAs were cloned and in vitro uptake assays of radio-labelled protein into isolated mitochondria indicated that 3 genes, NDA1, NDA2 and NDC1, encoded internal or matrix facing ND proteins, while 3 genes, NDB1, NDB2 and NDB4, encoded external or intermembrane space facing ND proteins. Analysis of expression of the various ND genes, along with other components of the mitochondrial electron transport chain suggested that NDA1 was expressed in a manner that suggested it played a role in

integrating mitochondrial and photosynthetic metabolism.

Overall, the expression of genes encoding ND proteins displayed different patterns. From these studies, it was concluded that this family of proteins had undergone neo-functionalisation based on the fact that the proteins were on different sides of the inner mitochondrial membrane and thus are proposed to serve different roles. In addition, sub-functionalisation was also observed, as differential expression of genes encoding protein isoforms of ND proteins located on both the external and internal side of the inner membrane was observed. In the case of the PRAT family of transporters, 17 genes encode PRAT-like proteins in Arabidopsis. Analysis of the predicted proteins revealed that this family had undergone significant divergence in plants compared to other organisms with proteins ranging in size from 128 to 261 amino acids. In vitro uptake assays, in vivo GFP localisations and Western blotting indicated that 10 of the 17 genes in Arabidopsis encode proteins targeted to mitochondria. The others encode outer envelope proteins of the chloroplast (OEP), similar to OEP16 that was first characterised in pea. Of these 10 proteins, 3 encode Tim17 like proteins, 3 encode Tim23 like proteins and 2 encode Tim22 like proteins. A protein encoded at the locus At2g42210 is found in Complex I (Meyer et al., 2008). The function of a PRAT protein encoded at the chromosomal locus At3g25120 is unknown...

Thesis (Ph.D.)--University of Western Australia, 2010

http://repository.uwa.edu.au:80/R/-?func=dbin-jump-full&object_id=29959&silo_library=GEN01