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 vital roles in the biogenesis of mitochondria. Two essential, highly conserved mitochondrial processes that depend upon the function of Hsp70 and J-protein molecular chaperones will be studied - translocation of proteins from the cytosol into the mitochondrial matrix and the generation of Fe/S clusters, critical co-factors for numerous enzymes. The goal of this proposal is to not only gain a better understanding of these two essential processes, but also to understand the mechanisms by which the action of Hsp70s and J-proteins are co-opted to make them competent to efficiently carry out specific roles in diverse biological processes. These two systems represent examples of the two basic features known to govern the ability of Hsp70s/J- proteins to function in diverse biological processes: localization to sites of action (protein import) and restricted client binding, rather than the promiscuous binding characteristic of function in protein folding (Fe/S cluster biogenesis). 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, and its obligate J-protein co-chaperone, Pam18, at its core. Genetic, biochemical and structural approaches will be used, with a goal of understanding the regulated protein:protein interactions that have evolved to drive efficient translocation of proteins across the membrane. The results generated will also provide a framework for understanding Hsp70/J-protein machines in other systems, as mechanisms such as tethering regulated by interaction with client proteins are likely common regulatory strategies in chaperone systems. 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, via action of an "assembly complex" prior to transfer to recipient apo-proteins. The specialized J-protein:Hsp70 chaperone pair, Jac1:Ssq1, facilitates transfer of the cluster. Using biochemical interaction assays and exploiting mutant proteins having defects in their interactions with partner proteins, the mechanism by which binding of Jac1 initiates the transfer process will be studied to understand the kinetic switch between cluster formation and cluster transfer. Our work on this system will also serve as a model for a more general understanding of the means by which molecular chaperones modulate protein:protein interactions in other biolgical processes.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM027870-34A1
Application #
8631380
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Wehrle, Janna P
Project Start
1980-04-01
Project End
2018-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
34
Fiscal Year
2014
Total Cost
$348,672
Indirect Cost
$105,649
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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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