The emerging evidence supports the proposition that the mitochondrial respiratory chain functions via organized multicomplex structures called supercomplexes. Complex I assembly plays a paramount role in the assembly of supercomplexes. However, our understanding of Complex I function and its regulation is incomplete. The Complex I assembly process, especially the details involving mtDNA-encoded subunits, is largely unclear. In particular, only a very limited number of assembly factors have been identified. Our long term goal is to understand the dynamics of mitochondrial respiratory machinery, including their assembly and turnover processes. The objective of this particular application is to understand the role of several key players in Complex I assembly including several mtDNA-encoded subunits: ND4, ND5 and ND6 and putative Complex I assembly factors DsbA-L and HSP60. The study of Complex I assembly has been difficult since the conventional model S. cerevisiae does not have Complex I and it is almost impossible to induce specific mutations in mammalian mtDNA;thus, mutant cells carrying mtDNA mutations in genes encoding Complex I subunits are rare. We have previously established an efficient method to isolate cells carrying mtDNA mutations and generated several cell models with Complex I assembly deficiency which were then used to initiate comprehensive studies on this complex. We then isolated several cell lines, derived from these mutant lines carrying mtDNA mutations, that had restored Complex I assembly. Further characterizations of these cell lines employing both molecular and proteomics approaches have implicated molecular chaperones HSP60 and DsbA-L in Complex I and supercomplex assembly. The central hypothesis for this application is that the assembly of respiratory Complex I is a delicately regulated process in which the mtDNA-encoded subunits ND4, ND5 and ND6, and assembly factors DsbA-L and HSP60 play distinct roles. To test this hypothesis, we propose to pursue the following three specific aims: 1) Determine the role of mtDNA-encoded subunits ND4, ND5 and ND6 in Complex I dynamics;by combining pulse-chase and BNG analysis, we will follow the step- wise assembly of Complex I and supercomplexes. 2) Characterize the role of HSP60 in Complex I and supercomplex assembly;we will test the ability of HSP60 to suppress some Complex I assembly defects using over-expression approach. 3) Characterize the role of DsbA-L in Complex I assembly and characterize the mouse model with neuronal specific knockout DsbA-L by taking advantage of our collaborators'established animal model. The approach is innovative, because it combines our unique cell and animal models with newly developed analytic methods to understand the complexity of the respiratory complex assembly process. The research is significant, because elucidating these mechanisms could provide new insight into the pathogenesis of diseases resulting from mitochondrial Complex I deficiency. In addition, we anticipate identification of novel risk genes involved in human diseases associated with mitochondrial dysfunction.

Public Health Relevance

The proposed research is relevant to public health because the studies on respiratory Complex I assembly will shed light on the pathogenesis of many human disorders with Complex I dysfunction. It would also identify new candidate genes for neurological diseases and provide new treatment targets for mitochondrial diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM109434-01A1
Application #
8762078
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Anderson, Vernon
Project Start
2014-08-01
Project End
2018-05-31
Budget Start
2014-08-01
Budget End
2015-05-31
Support Year
1
Fiscal Year
2014
Total Cost
$284,050
Indirect Cost
$94,050
Name
University of Texas Health Science Center
Department
Biology
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Vartak, Rasika S; Semwal, Manpreet Kaur; Bai, Yidong (2014) An update on complex I assembly: the assembly of players. J Bioenerg Biomembr 46:323-8