One hundred and two clinical phenotypes have been associated with mutations in a quarter of the known 406 nuclear-encoded human mitochondrial proteins. Estimated 500-600 nuclear encoded mitochondrial proteins await identification. The physiological mechanisms operating in mitochondria are highly conserved among eukaryotes. Hence, insights gained into the function of mitochondrial proteins in yeast can be correlated to their orthologues in human that are involved in health and disease. Using single-gene yeast deletion mutants, 265 novel proteins with a respiratory deficiency have been identified. By blasting these proteins against an expressed sequence tag library and, consequently, correlating them to mapped putative mitochondrial disorders, candidate genes could be identified for such mitochondrial disorders as optic atrophy 4, Moebius syndrome 2, Friedreich ataxia 2, and thyroid cancer with cell oxyphilia. As a follow-up, a complementary study of mitochondrial function at the transcriptional and translational level is proposed. Using Fourier transform ion cyclotron resonance mass spectrometry of tryptic digests of isolated yeast mitochondria, accurate mass tags for yeast nuclear encoded mitochondrial proteins will be obtained with the goal to identify all mitochondria located proteins. High-density DNA array expression analysis of proteins identified by gene deletion and the proteomics project will be used to identify pathway specific mRNA signature profiles, which can be used to group unclassified genes into an operational network. Thirty-two yeast deletion strains involved in the function of the respiratory chain, amino acid metabolism, heme biosyntesis, and membrane transport were selected under the criteria of their quantitative deletion phenotype and a human orthologue involved in a mitochondrial disorder. In addition, to study more subtle alterations of cellular and mitochondrial function, experiments using the yeast deletion collection on several iron and thiamin conditions are proposed. Further, mRNA signature profiling will be conducted on human cell lines established from patients with mitochondriopathies. These cell lines are related to yeast, 19 have a known gene mutation and a yeast orthologue involved in the similar pathway and partially found with a respiratory deficiency. In a pilot project, a library of accurate mass tags for human mitochondrial proteins will be generated for the characterization of human cell lines derived from patients with known or putative mitochondriopathies. The concordance of protein data with those obtained by means of human mRNA expression arrays and the functional genomic findings from yeast will be assessed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063883-03
Application #
6894299
Study Section
Genome Study Section (GNM)
Program Officer
Poodry, Clifton A
Project Start
2003-06-01
Project End
2006-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
3
Fiscal Year
2005
Total Cost
$666,264
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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