Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

No description available.

Both resistance and endurance exercise elicit an increase in muscle protein synthesis during recovery from exercise. Ingestion of amino acids augments the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Our work showed that 8 wk of unilateral resistance training induced muscle hypertrophy only in the exercised limb. Importantly, using this unilateral model we showed that muscle hypertrophy was confined to the exercised leg and occurred without measurable changes in circulating anabolic hormones. We then went on to use the unilateral leg resistance exercise model to study how animal-derived (milk) and plant-derived (soy) proteins impacted acute post-exercise protein turnover. We observed that ingestion of soy or milk protein resulted in a positive net protein balance following resistance exercise. Moreover, milk promoted a greater net protein balance and muscle protein synthesis than soy protein. In the final study, a key finding was that acute endurance and resistance exercise differentially stimulated myofibrillar and mitochondrial protein synthesis and also differentially affected cellular signaling proteins involved in the regulation of the protein synthetic response. Specifically, the acute, untrained state response showed that resistance exercise stimulated myofibrillar and mitochondrial protein synthesis while endurance exercise stimulated mitochondrial protein synthesis. Following resistance training only myofibrillar protein synthesis increased after exercise, while mitochondrial protein synthesis was unchanged. Endurance exercise training did not affect the acute protein synthetic response and so following training mitochondrial protein synthesis was stimulated as it was acutely, prior to training. In conclusion, the studies within this thesis provided novel insights on the impact of intact dietary proteins and differing modes of exercise on the control skeletal muscle protein metabolism. / Thesis / Doctor of Philosophy (PhD)

No description available.

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

<p> Two studies are presented in this thesis. First part is about the origin of tRNA genes in Trypanosoma and Leishmania. These organisms have special mitochondrial DNA, termed kinetoplast DNA (kDNA), which is unique in its structure and function. kDNA is a massive network which is composed of thousands of connected DNA circles. Unlike most other mitochondrial genomes, there is no gene encoding tRNAs in their kDNAs. So all the tRNAs used in mitochondria must be encoded on nuclear genes and transported from the cytoplasm into the mitochondria. So our question of interest is where the tRNA genes in their nucleus come from. We carry out phylogenetic analysis of these genes and the corresponding ones in bacteria, mitochondria and eukaryotic nuclei. There is no evidence indicating gene transfer from mitochondria to nucleus on the basis of this analysis. These results are consistent with the simplest hypothesis, i.e. that all tRNA genes of Trypanosoma and Leishmania have the same origin as nuclear genes of other eukaryotes.</p> <p> The second part is about the comparison of eukaryote phylogenies obtained from mitochondrial rRNA and protein sequences. We carried out phylogenetic analysis for the species which have complete mitochondrial genomes by using both concatenated mitochondrial rRNA and protein sequences. We got phylogenies for three groups, fungi/metazoan, plant/algae and stramenopile/alveolate group. The analysis is useful for the further study of position of the genetic code changes and the mechanisms involved.</p> / Thesis / Master of Science (MSc)

Made available in DSpace on 2015-03-26T13:36:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 318019 bytes, checksum: f14201c1f1fe349bcc7b8771be868f8e (MD5) Previous issue date: 2006-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The effects of aluminum on growth, respiratory activities and on some enzymes related to reactive oxygen species (ROS) elimination were examined in roots of two corn seedlings cultivated in nutrient solution, by comparing a susceptible (BR 106) and a tolerant cultivar (BR 206). Growth of the main root decreased in both cultivars, mainly in the susceptible one, at aluminum concentration of 50 and 100 µM. Only in the tolerant cultivar aluminum induced a decrease in the leak of electrolytes. On the contrary, the susceptible cultivar showed significant increase in lipid peroxidation. Aluminum promoted higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), only in the tolerant cultivar. No difference was observed in the activities of catalase (CAT), peroxidases (POX) and glutathione reductase (GR) in both cultivars. Ascorbate content was not altered, but the ascorbate redox state (ratio ascorbate/dehydroascorbate) increased in the tolerant cultivar. In absence of aluminum, respiratory oxygen consumption was higher in the tolerant cultivar, both in states 3 and 4. These respiratory activities were decreased by aluminum in the susceptible cultivar, but were increased in the tolerant one. ADP/O ratios were decreased by aluminum, in both cultivars at a similar extent. The cytochrome c pathway was not altered by aluminum in the tolerant cultivar, but decreased in the susceptible one. Without aluminum, the susceptible cultivar showed a higher alternative oxidase (AOX) activity than the tolerant one. Aluminum promoted a decrease in AOX activity in the susceptible cultivar, the opposite response being observed in the tolerant one. In both cultivars aluminum induced an increase above 120% in the residual oxygen consumption. Activity of the plant uncoupling mitochondrial protein (PUMP) was decreased by aluminum in the susceptible cultivar and enhanced in the tolerant one. These results suggest that the tolerant cultivar possesses a more efficient enzymatic system for ROS removal than the sensitive one. Furthermore, the partial mitochondrial uncoupling, resulting from higher AOX and PUMP activities should have contributed to the higher aluminum stress tolerance showed by cultivar BR 206. / Os efeitos do alumínio sobre o crescimento, a respiração e as atividades de algumas enzimas envolvidas na eliminação de espécies reativas de oxigênio foram avaliadas em plântulas de duas cultivares de milho, uma sensível (BR 106) e outra tolerante ao alumínio (BR 206), cultivadas em solução nutritiva, pH 4,0. O alumínio, nas concentrações de 50 e 100 µM, reduziu o crescimento da raiz principal das duas cultivares, especialmente da cultivar sensível. O alumínio reduziu o extravasamento de eletrólitos, apenas nas raízes da cultivar tolerante e aumentou a peroxidação de lipídios, apenas na cultivar sensível. O tratamento com alumínio resultou em aumentos significativos nas atividades da dismutase do superóxido (SOD) e da peroxidase do ascorbato (APX), apenas na cultivar tolerante. Entretanto, não foram observadas diferenças significativas nas atividades da catalase (CAT), das peroxidades (POX) e da redutase da glutationa (GR), em ambas as cultivares. Os teores de ascorbato também não apresentaram variação significativa, mas foi observado aumento no estado redox (razão ascorbato/desidroascorbato), porém apenas na cultivar tolerante. No tratamento controle, as taxas respiratórias de mitocôndrias isoladas da cultivar tolerante se apresentaram mais elevadas, tanto no estado 3 como no estado 4. Estas taxas foram reduzidas pelo alumínio na cultivar sensível, e aumentadas na cultivar tolerante. As razões ADP/O foram reduzidas pelo tratamento com alumínio, nas duas cultivares, na mesma proporção. A rota do citocromo c não foi alterada pelo alumínio, na cultivar tolerante, mas foi reduzida na cultivar sensível. Na ausência de alumínio, a cultivar sensível apresentou atividade da oxidase alternativa (AOX) mais elevada, que foi reduzida pela presença de alumínio. Ao contrário, o consumo de oxigênio pela rota alternativa foi aumentado, pelo alumínio, na cultivar tolerante. Nas duas cultivares, o alumínio promoveu acréscimos superiores a 120% no consumo residual de oxigênio. A atividade da proteína desacopladora de plantas (PUMP) foi diminuída pelo alumínio, na cultivar sensível, e aumentada, na tolerante. Estes resultados sugerem que a cultivar tolerante possua um mecanismo enzimático mais eficiente de remoção ou neutralização de espécies reativas de oxigênio que a cultivar sensível. Além disso, o parcial desacoplamento mitocondrial observado, resultante do aumento das atividades da oxidase alternativa e da proteína desacopladora, deve contribuir para a maior tolerância da cultivar BR 206 ao estresse por alumínio.

Contrairement à la vision classique des ARNms eucaryotes qui ne contiendrait qu’une seule séquence codante, de nombreuses évidences expérimentales montrent que ces ARNms contiennent plusieurs séquences codantes qui permettraient l’expression de plusieurs protéines différentes. Les ARNms sont donc multicodants et contiennent des cadres de lectures alternatifs (AltORFs pour alternative open reading frames). Ces ORFs alternatifs sont présents dans les régions non-traduites (UTRs) ou chevauchant le RefORF (cadre de lecture ouvert de référence) dans les cadres de lectures non-canoniques +2 et +3. Le protéome est donc plus complexe que ce que l’on pense. Toutefois, le rôle et la fonction de ces nouvelles protéines restent à être investigués. Au cours de mon projet de recherche à la maîtrise, j’ai commencé la caractérisation de la protéine alternative AltMiD51, codée dans le 5’UTR du gène bicistronique MIEF1/SMCR7L/MID51 et co-exprimée avec sa protéine de référence MiD51. Par des approches variées de biologie moléculaire, cellulaire et biochimique, j’ai d’abord démontré et confirmé la localisation cellulaire de la protéine AltMiD51 à la mitochondrie. Par la suite, j’ai pu démontrer que la présence d’AltMiD51 affecte significativement la morphologie mitochondriale en fragmentant celle-ci. De plus, j’ai pu davantage cibler la région qui contient l’information de sa localisation ainsi que son effet de fragmentation, soit seulement les 23 premiers acides aminés de sa séquence. J’ai également observé que cette région Nterminale (a.a.23) est encore plus efficace pour induire la fragmentation des mitochondries. J’ai pu démontrer que le motif protéique L-Y-R est essentiel pour l’activité de fragmentation. J’ai également validé l’interaction in vivo de AltMID51 avec sa protéine partenaire ACPM (Acyl carrier protein) dans des foci mitochondriaux. En conclusion, mes travaux à la maîtrise ont permis de mettre en évidence que la protéine AltMiD51 est un nouveau facteur impliqué dans la fission mitochondriale. Ces résultats ouvrent de nouvelles perspectives en ce qui concerne la caractérisation de nouvelles protéines alternatives et par leur contribution dans la biologie moléculaire de la cellule. / Abstract : Challenging the dogma that eukaryotic mRNAs contain a single coding sequence, an ever-growing number of studies highlight the possibility for these mRNAs to have several coding sequences, and thereby code for multiple proteins. Thus, eukaryotic mRNAs are multicoding and present alternative open reading frames (AltORFs). These alternative ORFs are present in the non-translated region (UTRs), and within or overlapping the RefORF (reference open reading frame) in non-canonical frames (+2 and +3). The proteome is indeed more complex than we initially thought. However, the role and biological function of these proteins remain to be elucidated. Over the course of my MPhil, I started characterizing the alternative protein AltMiD51, encoded in the 5’UTR of the bicistronic MIEF1/SMCR7L/MID51 gene, and coexpressed with its reference protein (MiD51). Using a wide range of molecular biology, cellular and biochemistry assays, I first demonstrated AltMiD mitochondrial localisation. I then proved AltMiD51 expression alters mitochondrial dynamics, enhancing a fragmented morphology. Moreover, I further characterized the sequence region responsible for AltMiD51 localisation and mitochondrial fragmentation, namely the first 23 amino acids. I also observed that this N-terminal region alone presents a stronger phenotype of mitochondrial fragmentation. In addition, I proved the L-Y-R domain is essential for AltMiD51 fragmentation activity, and I validated AltMiD51 in vivo interaction with ACPM (Acyl Carrier Mitochondrial Protein) within mitochondrial foci. Eventually, the work presented here highlighted AltMiD51 as a novel factor involved in mitochondrial fragmentation. These results shed light on new perspectives regarding the characterisation of alternative proteins and their contribution to the cellular metabolism.

Most mitochondrial proteins are encoded by nuclear DNA, and synthesised in the cytosol, then imported into the different mitochondrial subcompartments. To reach their destination, mitochondrial inner membrane proteins require import across the outer mitochondrial membrane, and through the intermembrane space. This passage through the IMS is assisted by the small Tim proteins. This family is characterised by conserved cysteine residues arranged in a twin CX3C motif. They can form Tim9-Tim10 and Tim8-Tim13 complexes, while Tim12 appears to form part of a Tim9-Tim10-Tim12 complex that is associated with the inner membrane translocase TIM22 complex. Current models suggest that the biogenesis of small Tim proteins and their assembly into complexes is dependent on the redox states of the proteins. However, the role of the conserved cysteine residues, and the disulphide bonds formed by them, in small Tim biogenesis and complex formation is not clear. As there is no research about the structural characterisation of Tim12 and double cysteine mutants of Tim9, purification of these proteins was attempted using different methods. To investigate how cysteine mutants affect complex formation, the purified double cysteine mutants of Tim9 were studied using in vitro methods. It showed that the double cysteine mutants were partially folded, and they can form complexes with Tim10 with low affinities, suggesting disulphide bonds are important for the structures and complex formation of small Tim proteins. The effect of cysteine mutants on mitochondrial function was addressed using in vivo methods. It showed that cysteines of small Tim proteins were not equally essential for cell viability, and growth defect of the lethal cysteine mutant was caused by low level of protein. Thus, the conclusion of this study is that disulphide bond formation is highly important for correct Tim9- Tim10 complex formation, and yeast can survive with low levels of complex, but it results in instability of the individual proteins.