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.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.