Final Activity Report Summary - MITHOM (Investigating the core function of mitochondrial homologues)
Mitochondria are vital eukaryotic organelles, but very little is known about their structure and functions outside of a few well-studied model species. It has recently been shown that the diversity of mitochondrial form and function is greater than previously realised. Parasitic eukaryotes including the important human parasites Entamoeba, Giardia, Trichomonas and microsporidia, previously thought to be without mitochondria, have now been shown to retain organelles of mitochondrial ancestry. These discoveries suggest that all eukaryotes contain a mitochondrial homologue and also suggest that there may be an essential function that makes these organelles vital for eukaryotic cells. It has recently been hypothesised that the core function of mitochondrial homologues is the biosynthesis of iron-sulfur (FeS) clusters for insertion into mitochondrial and cytosolic FeS cluster containing proteins. The Entamoeba mitosome is one of the simplest mitochondrial homologues making it an ideal model system to identify any common core function.
To generate hypotheses of organelle function I carried out comparative genomic analyses to identify the core set of proteins for all mitochondrial homologues. The genomes analysed included species with different types of mitochondrial homologues including Encephalitozoon, Entamoeba and Giardia (all with mitosomes), Trichomonas (hydrogenosomes) and parasites with highly reduced mitochondria (e.g. Cryptosporium, Plasmodium). Two possible mitosomal proteins from Entamoeba histolytica and one known protein were selected for immunolocalisation and functional studies. The proteins were, Cpn60 - a mitochondrial chaperonin with a known localisation to the E. histolytica mitosome as a positive control. A second mitochondrial chaperonin Hsp70 was previously shown to co-localise with Cpn60 in mitosome enriched sub-cellular fractions, but so far has not been directly localised to the E. histolytica mitosome. The third protein is the pyridine nucleotide transhydrogenase (PNT). The PNT has been shown in classical mitochondria to be regulated by a proton gradient and can in some contexts generate the proton gradient, which is a feature of mitochondrial inner membranes crucial for yeast FeS cluster formation.
Two complementary approaches were chosen to investigate the cellular localisation of the candidate mitosomal proteins. In the first approach, I made specific antisera to the Entamoeba recombinant protein PNT. I also generated anti-Cpn60 antisera to complement the existing anti Cpn60 antisera we received from our collaborator Dr Clark. Both proteins were expressed as recombinant proteins in E. coli and the purified proteins were used to make antisera in rabbits (Cpn60) or chickens (PNT). These antisera will be available shortly and will be tested for specificity for subsequent use in co-localisation experiments and Western blot analyses on subcellular fractions including mitosomal enriched sub-cellular preparations. The second approach was based on homologous transfection followed by confocal indirect immuno-fluorescence analyses (IFA) to visualise the proteins within the Entamoeba cell. For this approach Hsp70, Cpn60 and PNT were epitope tagged with a c-myc tag at their C-terminus and introduced into two different Entamoeba specific expression vectors mediating (i) constitutive protein expression or (ii) tetracycline regulated protein expression. Entamoeba cells were transfected with the different vectors and the preliminary results shows IFA signals for PNT-myc within the Entamoeba cells, and its partiall co-localisation with Cpn60. Further experiments are needed to verify the specificity of the signal with co-localisation of PNT-myc expressing cells with the newly raised antisera against Cpn60 (mitosomal protein) and for the complementary experiment with Cpn60-myc expressing cells co-labelled with the newly raised antisera raised against PNT.
To generate hypotheses of organelle function I carried out comparative genomic analyses to identify the core set of proteins for all mitochondrial homologues. The genomes analysed included species with different types of mitochondrial homologues including Encephalitozoon, Entamoeba and Giardia (all with mitosomes), Trichomonas (hydrogenosomes) and parasites with highly reduced mitochondria (e.g. Cryptosporium, Plasmodium). Two possible mitosomal proteins from Entamoeba histolytica and one known protein were selected for immunolocalisation and functional studies. The proteins were, Cpn60 - a mitochondrial chaperonin with a known localisation to the E. histolytica mitosome as a positive control. A second mitochondrial chaperonin Hsp70 was previously shown to co-localise with Cpn60 in mitosome enriched sub-cellular fractions, but so far has not been directly localised to the E. histolytica mitosome. The third protein is the pyridine nucleotide transhydrogenase (PNT). The PNT has been shown in classical mitochondria to be regulated by a proton gradient and can in some contexts generate the proton gradient, which is a feature of mitochondrial inner membranes crucial for yeast FeS cluster formation.
Two complementary approaches were chosen to investigate the cellular localisation of the candidate mitosomal proteins. In the first approach, I made specific antisera to the Entamoeba recombinant protein PNT. I also generated anti-Cpn60 antisera to complement the existing anti Cpn60 antisera we received from our collaborator Dr Clark. Both proteins were expressed as recombinant proteins in E. coli and the purified proteins were used to make antisera in rabbits (Cpn60) or chickens (PNT). These antisera will be available shortly and will be tested for specificity for subsequent use in co-localisation experiments and Western blot analyses on subcellular fractions including mitosomal enriched sub-cellular preparations. The second approach was based on homologous transfection followed by confocal indirect immuno-fluorescence analyses (IFA) to visualise the proteins within the Entamoeba cell. For this approach Hsp70, Cpn60 and PNT were epitope tagged with a c-myc tag at their C-terminus and introduced into two different Entamoeba specific expression vectors mediating (i) constitutive protein expression or (ii) tetracycline regulated protein expression. Entamoeba cells were transfected with the different vectors and the preliminary results shows IFA signals for PNT-myc within the Entamoeba cells, and its partiall co-localisation with Cpn60. Further experiments are needed to verify the specificity of the signal with co-localisation of PNT-myc expressing cells with the newly raised antisera against Cpn60 (mitosomal protein) and for the complementary experiment with Cpn60-myc expressing cells co-labelled with the newly raised antisera raised against PNT.