Mitochondria play a central role in oxidative metabolism of eukaryotic cells by providing most of the ATP needed for their survival. Maintenance of this vital function depends on a modest number of mitochondrial genes and a much larger pool of genetic information residing in the nucleus. We have continued to use respiratory deficient mutants of the unicellular yeast Saccharomyces cerevisiae to study the mechanisms by which the H+ translocating ATPase (F1-F0 ATPase), the bc1 complex and mitochondrial ribosomes are assembled. In the coming period we intend to continue three areas of studies. First, we will extend our functional analyses of Atp25p and Atp23p aimed at better understanding the manner in which these chaperones promote, respectively, formation of the ATPase subunit 9 ring and its interaction with subunit 6. To characterize the intermediates and the events leading to assembly of the ATPase, we will exploit recently obtained strain constructs in which the mitochondrial genes for subunits 6, 8, and 9 of the F0 sector are expressed with tags suitable for detection and affinity purification. We will also continue to screen for other ATPase assembly genes, of which we believe more remain to be found. Another important area of studies is to explore how the AE complex functions in expression of subunits 6 and 9 and to test its possible role in docking mitochondrial ribosomes to regions of the inner membrane where these subunits assemble into the holoenzyme.
The second aim of this proposal is related to our recent discovery that translation of subunit 6 of F0 depends is activated by the F1 ATPase. The immediate questions addressed are: 1) Is translational activation of the subunit 6 mRNA dependent on a specific subunit of F1;2) is there a sequence element in the RNA that mediates translational activation by F1 and;3) does activation depend on the interaction of F1, or a subunit thereof, with the mRNA? The third aim is to clarify the role of CPB3 in the stability and/or membrane insertion of mitochondrially encoded cytochrome b, an important catalytic subunit of the bc1 complex. Most of the yeast genes we have studied in the past have homologues in higher eukaryotes and an increasing number have been shown to be involved in human neuromyopathies. The studies described in this proposal will continue to improve the yeast model as it applies to human mitochondrial diseases.

Public Health Relevance

Because of its experimental tractability, the unicellular yeast Saccharomyces cerevisiae has become a useful model for unraveling the genetic basis of human neuromyopathies stemming from lesions in mitochondria. One of our current goals is to exploit respiratory deficient mutants of this organism to understand the mechanism by which mitochondrial ATPase (ATP synthase), which plays a central role in energy metabolism, is assembled and how this process is regulated. A second goal is to continue clarifying the functions of accessory protein factors that are essential for assembly of the ATPase and of the bc1 complex, another important member of the terminal respiratory pathway.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL022174-33
Application #
8616774
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Wong, Renee P
Project Start
1977-12-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
33
Fiscal Year
2014
Total Cost
$289,800
Indirect Cost
$109,800
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
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