In the past decade, mitochondrial fusion and fission have emerged as major regulators of mitochondrial function. Defects in these processes are associated with human disease, and mouse studies indicate their importance in maintaining a healthy mitochondrial population within cells. This proposal focuses on the mechanism of mitochondrial fission, which is implicated in control of mitochondrial morphology, degradation of mitochondria by autophagy, control of mitochondrial distribution, and regulation of apoptosis. During the process of mitochondrial fission, Drp1 (dynamin-related protein 1) is recruited from the cytosol onto the mitochondrial surface. Here it assembles into an oligomeric complex that wraps around the mitochondrial tubule and constricts it to mediate membrane scission. In order for Drp1 to be recruited to mitochondria, the mitochondrial outer membrane contains four Drp1 receptors: Fis1, Mff, MiD49, and MiD51. Cellular studies indicate that the latter three molecules are the most functionally important for mitochondrial fission. This proposal focuses on obtaining a structural, biochemical, and cellular understanding of how these molecules mediate Drp1 recruitment.
In Aim 1, X-ray crystallography is used to obtain atomic structures of Mff, MiD49, and MiD51. Current work has already yielded a high-resolution structure of MiD51. Surprisingly, this structure indicates that MiD51 has a nucleotidyl transferase domain, with conservation of key residues that are implicated in nucleotide binding and catalysis. Additional structures of MiD51 in complex with nucleotide will be obtained.
In Aim 2, biochemical studies are used to advance the understanding of MiD51 and MiD49 function. Given that MiD51 has a nucleotidyl transferase fold, the nucleotide binding specificity of MiD51 will be measured with quantitative binding assays. In addition, enzymatic assays will be used to determine if MiD51 has a catalytic function in nucleotide transfer. Similar studies will be done with MiD49, which is homologous to MiD51.
In Aim 3, structure-function analysis in cell culture is used to determine whether nucleotide binding, catalysis, or dimerization is important for the ability of MiD51 to recruit Drp and mediate fission. MiD51 and MiD49 knockout cells for further structure-function analysis will be generated through the use of genome editing technology. Finally, a genetic approach will be utilized to map the Drp1 binding site on Mff, MiD49, and MiD51. Taken together, these studies will greatly advance the understanding of the structural biology of Drp1 receptors and may ultimately lead to new methods to modulate mitochondrial fission. 1

Public Health Relevance

Mitochondria are crucial organelles that generate energy for cells, and their dysfunction underlies many human diseases. Our studies will reveal the molecular mechanism of mitochondrial division, a process that is essential for mitochondrial function. This information may lead to new methods to control mitochondrial division, an advance that can benefit human diseases associated with mitochondrial dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM110039-01
Application #
8670204
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2014-04-01
Project End
2017-12-31
Budget Start
2014-04-01
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$361,305
Indirect Cost
$144,305
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Mishra, Prashant; Chan, David C (2014) Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol 15:634-46
Carelli, Valerio; Chan, David C (2014) Mitochondrial DNA: impacting central and peripheral nervous systems. Neuron 84:1126-42