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Gawryluk, R.M.R3., Spencer, D.F3., Smith, D.G.S.2, Pearlman, R.E.1, Siu, K.W.M.2, and Gray, M. W.3 The mitochondrial energy cycle of the ciliate protozoon, Tetrahymena thermophila; a proteomic analysis Departments of Biology1 and Chemistry2, York University, Toronto, Canada and Department of Biochemistry, Dalhousie University, Halifax, Canada3 Comparative studies of mitochondrion-encoded genes demonstrate that the mitochondrial genome had an α-proteobacterial origin. However, the vast majority of mitochondrial proteins are now encoded in nuclear DNA. These genes are transcribed in the nucleus, translated on cytosolic ribosomes and directed to mitochondria by N-terminal protein extensions known as targeting peptides. Since most mitochondrial proteins are nucleus-encoded, proteomics-based approaches are crucial to identifying the full mitochondrial protein complement (‘mitochondrial proteome’) and elucidating the function and evolution of individual protein components. Previous mitochondrial proteomics efforts have focused on fungi, animals and plants. These studies have neglected the unicellular protists, which comprise the bulk of eukaryotic biodiversity. We have used tandem liquid chromatography-mass spectrometry (LC/LC-MS/MS) to analyze purified mitochondria from Tetrahymena thermophila, identifying 545 nucleus-encoded and 29 mitochondrion-encoded proteins, the first systematic characterization of a protistan mitochondrial proteome. Mitochondria are vital to many eukaryotic cellular processes, including apoptosis, lipid and amino acid metabolism, and iron homeostasis. However, they are probably best known for their role in cellular energy production. Herein, we detail the components of key energygenerating processes associated with Tetrahymena mitochondria: glycolysis, the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC). We utilize the 573 mitochondrial proteins that we have identified via MS to catalogue the key energy generating proteins associated with Tetrahymena mitochondria and complement our proteomics-based analysis with in silico similarity searches. The latter analysis provides information about the presence or absence of a corresponding coding sequence in the macronuclear or mitochondrial genomes of Tetrahymena. Our analysis indicates the following: glycolysis is associated with Tetrahymena mitochondria. It is unclear whether glycolytic enzymes are localized to the outer membrane of mitochondria for increased metabolic efficiency, or whether some enzymes perform novel (intra-) mitochondrial functions in ciliates; the TCA cycle of Tetrahymena is well conserved, although gene loss, gene duplications and protein re-targeting have complicated its evolutionary history; many of the core electron transport chain subunits were detected in ciliates by MS or bioinformatics, but Tetrahymena’s ETC may be missing several components, such as SdhC, SdhD and Cox3 (are these components simply divergent proteins, or have they been lost/replaced?); analysis of isolated ETC complexes from other species demonstrates lineagespecific additions (e.g., the bacterial core of Complex I contains 14 subunits, whereas bovine Complex I contains 46); and our MS analysis identified a large number of small mitochondrial proteins specific to ciliates. Funding to MWG and to REP from CIHR and to KWMS and REP from the NSERCand Eli Lilly Canada’s CRD Grants is acknowledged. Infrastructure support to KWMS from the Ontario Research and Development Challenge Funds and MDS SCIEX is acknowledged. RMRG thanks Killam Trusts and NSERC for funding. Gawryluk, R.M.R3., Spencer, D.F3., Smith, D.G.S.2, Pearlman, R.E.1, Siu, K.W.M.2, and Gray, M. W.3 An analysis of mitochondrial protein targeting and import in the ciliate protozoon, Tetrahymena thermophila. Departments of Biology1 and Chemistry2, York University, Toronto, Canada and Department of Biochemistry, Dalhousie University, Halifax, Canada3 Mitochondria are organelles involved in a wide variety of eukaryotic cellular processes, including apoptosis, iron homeostasis and oxidative phosphorylation. Comparative studies of mitochondria-encoded genes demonstrate that the mitochondrial genome had an α-proteobacterial origin. Although mitochondria possess a genome, most mitochondrial proteins are nucleusencoded. These proteins are directed to mitochondria via targeting peptides (mTPs), N-terminal extensions typically rich in basic and hydroxylated amino acids and capable of forming amphiphilic α-helices with basic and hydrophobic faces. Target peptides are recognized at the outer mitochondrial membrane and imported via multiprotein complexes known as the translocases of the outer and inner mitochondrial membranes (TOM and TIM complexes, respectively). These complexes appear to be largely eukaryotic inventions, as few subunits have identifiable prokaryotic homologues. Because most mitochondrial proteins are nucleus-encoded, proteomics-based approaches are crucial to identifying the full mitochondrial protein complement (‘mitochondrial proteome’) and elucidating the function and evolution of individual protein components. Previous mitochondrial proteomics efforts have focused on fungi, animals and plants, and have neglected the unicellular protists, which comprise the bulk of eukaryotic biodiversity. We have used liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) to analyze purified mitochondria from Tetrahymena thermophila, identifying 573 mitochondrial proteins in this ciliated protozoon. Here we present an analysis of Tetrahymena mTP characteristics and an inventory of the TIM/TOM apparatus. We have utilized the sequences of mitochondrial proteins identified by mass spectrometry to characterize mitochondrial protein targeting in Tetrahymena and have assessed the mitochondrial protein import machinery in Tetrahymena using in silico-based similarity searches.We demonstrate that mTPs in Tetrahymena generally adhere to the rules governing mTPs of other eukaryotes. Thus, it may be feasible to develop new, or utilize existing, bioinformatics-based techniques to further define Tetrahymena’s mitochondrial proteome based on target peptides. However, many proteins specific to ciliates do not possess typical mTPs. It would not have been possible to identify these as mitochondrial proteins by in silico methods alone, underscoring the importance of our proteomics-based approach. Tetrahymena encodes homologues of many core yeast protein import subunits, suggesting an early eukaryotic origin for much of the mitochondrial protein import machinery. Homologues for receptor/accessory subunits are, however, not present in Tetrahymena. Presumably there are functional analogues of the corresponding yeast subunits in Tetrahymena, but defining these would require biochemical purification of the Tetrahymena complexes. Funding to MWG and to REP from CIHR and to KWMS and REP from the NSERCand Eli Lilly Canada’s CRD Grants is acknowledged. Infrastructure support to KWMS from the Ontario Research and Development Challenge Funds and MDS SCIEX is acknowledged. RMRG thanks Killam Trusts and NSERC for funding. Hongyan Li, Noah A. Fine, K.W. Michael Siu, and Ronald E. Pearlman Functional characterization of a human disease gene homolog, HEH2, in Tetrahymena thermophila Departments of Biology and Chemistry, York University, Toronto, Ontario, Canada The human KIAA1279 gene is important in the development of the enteric and central nervous system. It has been observed that homozygous nonsense mutations of this gene are associated with the Goldberg-Shprintzen syndrome, featuring phenotypes such as mental retardation, characteristic faces and foot anomalies. KIAA1279 was initially isolated from a human brain EST library and the function remains unidentified. We isolated a cDNA clone (~1.7kb), designated as HEH2, from a full length vegetative cDNA library of Tetrahymena thermophila. HEH2 encodes a predicated protein product of 571 amino acids, which share 29% sequence similarity with expect value of e-146 to the human KIAA1279 gene product. We have been pursuing molecular characterization and functional analysis of HEH2 with the aim of sheding light on exploring the human disease gene function, using Tetrahymena as an in vivo model system. cDNA and genomic sequences of HEH2 are identical, indicating that no introns occur in HEH2 coding region. Northern blot hybridization to whole cell mRNA shows abundant HEH2 transcription in vegetative cells, no expression in starved cells, and very low levels of expression in conjugating cells. Small peaks of expression are observed at 8 and 14 hours of conjugation. Localization by green fluorescent protein (GFP) tagging shows that Heh2p has a distinct localization pattern to basal body-like structures along ciliary rows and in the oral apparatus and primordial apparatus. To elucidate the role of HEH2 in T.thermophila, we disrupted the HEH2 gene with a neo3 inactivation cassette. Biolistic transformation was performed to introduce the linearized HEH2 disruption plasmid construct into Cu428 cells. Paromomycin (pm) resistant transformants were selected by growing in progressively increasing concentration of pm up to 3 mg/ml with cadmium chloride (1ug/ml). Southern blot analysis using a 3’ specific probe for the HEH2 gene was performed with HindIII digested genomic DNA isolated from four ∆HEH2 strains grown in 1mg/ml pm. In all strains, the intensity of the two wild type bands was reduced and a band resulting from neo integrated into the HEH2 locus, was present. None of the strains showed complete replacement of the wild-type alleles. Comparing the intensities of the transformants and wild type bands, more than 50% of the mac HEH2 genes were replaced in each transformant, indicating that the cells can survive with less than 50% of their mac HEH2 genes. This result signifies that HEH2 is an essential gene in T. thermophila. Analysis of these somatic transformants, demonstrated slow growth, arrest of cytokinesis and unusual motility. Analysis of growth rates revealed that the doubling time of the ∆HEH2 strains was approximately 1.5 to 2 times longer than that of wild-type controls. Abnormal growth, including torsion, breaks, fragmentation and monster cells, were all observed in the ΔHEH2 strains. Nearly immobile cells and spirally swimming deformed cells were also observed. We are currently analysing these phenotypes in both somatic and germ line knockout transformants. Functional analysis of HEH2 in T. thermophila as it relates to the disease gene phenotype of the human homologue will be discussed. This work was supported by grants from CIHR and the NSERRC CRD program with Eli Lilly Canada.