Mitochondria, the site of a variety of important metabolic processes, are essential organelles of eukaryotic organisms. Pathological effects of reduced bioenergetic capacity and altered iron metabolism caused by mitochondrial dysfunction are common in human populations. Molecular chaperones play a vital role in the biogenesis of mitochondria. The goal of this proposal is to understand the mechanism of action of Hsp70/J-protein molecular chaperones in two critical mitochondrial processes - translocation of proteins from the cytosol into the mitochondrial matrix and the generation of Fe/S clusters, critical co- factors for numerous enzymes. The vast majority of the hundreds of proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes. Thus, efficient import of proteins is critical for mitochondrial function. The import motor required for driving proteins across the inner membrane into the matrix is composed of 5 essential components, with the matrix Hsp70, Ssc1, at its core. We will concentrate on regulatory mechanisms that increase the efficiency of the import motor. We will use genetic, biochemical and structural approaches, with a goal of understanding the specialized regulated protein:protein interactions that have evolved to drive efficient translocation of proteins across the membrane. The mitochondrial matrix contains a set of essential proteins devoted to the biogenesis of Fe/S clusters. The clusters are assembled on the scaffold protein, Isu, prior to transfer to recipient apo- proteins. The J-protein:Hsp70 chaperone pair, Jac1:Ssq1, binds to Isu and facilitates transfer of the cluster. To understand the mechanism of this chaperone function in Fe/S cluster biogenesis the temporal set of interactions between Isu and other proteins required for Fe/S cluster formation and transfer will be determined, using biochemical interaction assays and exploiting mutant proteins having defects in their interactions with partner proteins. The yeast mitochondrial system will also be used as a model to understand the molecular basis of the specialization of Hsp70s. Knowledge gained as to the basis of the specialization of multiple Hsp70s of the mitochondrial matrix will serve as a paradigm for understanding how Hsp70s have evolved to function in an array of physiological processes in other cellular compartments particularly in the case of the less well-defined human Hsp70 family.
The research described in this proposal focuses on understanding fundamental aspects of mitochondrial function and biogenesis. Mitochondria are essential organelles that are vital for energy production. Reduced mitochondrial function has been linked to a wide array of health issues from age-related neurological and cardiovascular disease to early onset neuromuscular disorders.
|Ting, See-Yeun; Schilke, Brenda A; Hayashi, Masaya et al. (2014) Architecture of the TIM23 inner mitochondrial translocon and interactions with the matrix import motor. J Biol Chem 289:28689-96|
|Manicki, Mateusz; Majewska, Julia; Ciesielski, Szymon et al. (2014) Overlapping binding sites of the frataxin homologue assembly factor and the heat shock protein 70 transfer factor on the Isu iron-sulfur cluster scaffold protein. J Biol Chem 289:30268-78|
|Majewska, Julia; Ciesielski, Szymon J; Schilke, Brenda et al. (2013) Binding of the chaperone Jac1 protein and cysteine desulfurase Nfs1 to the iron-sulfur cluster scaffold Isu protein is mutually exclusive. J Biol Chem 288:29134-42|
|Ciesielski, Grzegorz L; Plotka, Magdalena; Manicki, Mateusz et al. (2013) Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA. Biochim Biophys Acta 1833:2233-43|
|Ciesielski, Szymon J; Schilke, Brenda A; Osipiuk, Jerzy et al. (2012) Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution. J Mol Biol 417:1-12|
|Schilke, Brenda A; Hayashi, Masaya; Craig, Elizabeth A (2012) Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae. Genetics 190:1341-53|
|Hayashi, Masaya; Schilke, Brenda; Marszalek, Jaroslaw et al. (2011) Ancient gene duplication provided a key molecular step for anaerobic growth of Baker's yeast. Mol Biol Evol 28:2005-17|
|Pais, June E; Schilke, Brenda; Craig, Elizabeth A (2011) Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor. Mol Biol Cell 22:4740-9|
|Correia, Ana R; Wang, Tao; Craig, Elizabeth A et al. (2010) Iron-binding activity in yeast frataxin entails a trade off with stability in the alpha1/beta1 acidic ridge region. Biochem J 426:197-203|
|Pukszta, Sebastian; Schilke, Brenda; Dutkiewicz, Rafal et al. (2010) Co-evolution-driven switch of J-protein specificity towards an Hsp70 partner. EMBO Rep 11:360-5|
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