Functional Genomics and Proteomics of Mitochondria
EMSL Project ID
2391
Abstract
Despite the progress made in deciphering the genetics of the more than one hundred known mitochondrial disease, many cases remain unexplained mostly due to the fact that only 40-50% of the presumed 700-1000 nuclear encoded mitochondrial proteins have been identified to date. However, recent technical advances that enable the functional study of entire genomes and proteomes should accelerate the identification of those genes. Moreover, mitochondrial genes and pathways are highly conserved across eukaryotes, and there is already an impressive list of yeast-human homologues associated with human mitochondrial disease. The Objective of the proposed research is to utilize the tremendous experimental resources available for the study of yeast, that have been partly pioneered in Dr. Davis? lab at Stanford University, in combination with the application of the advanced proteome analysis developed by Dr.Smith?s lab at EMSL/PNL, to elucidate new mitochondria proteins and pathways at an accelerated pace, which shall advance our understanding of mitochondria related human diseases. To achieve the above goal, the following specific aims are proposed (for details, please see attached mitochondria_proposal). Specific Aim 1: To identify new mitochondrial genes, the high throughput proteome analysis using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry will be applied to identify accurate mass tags for tryptic digested proteins of isolated yeast mitochondria. The sequences of the peptides analyzed will be searched against a database of known and candidate yeast mitochondria genes. Currently, 420 nuclear encoded genes have been located in yeast mitochondria. Besides, about 400 candidate genes are identified at Dr.Davis? lab, because yeast strains lacking each of these genes show defects in mitochondrial functions. The new proteins identified will be further verified by making constructs fused with Green Fluorescence Proteins (GFP) followed by in vivo localization assays. Gene expression analysis using DNA microarrays will be applied to elucidate the biological functions of these genes. Specific Aim 2: To facilitate research on human mitochondriopathies, we will identify accurate mass tags for human mitochondrial proteins. Candidate genes for database searches will be generated by blasting the results from Specific Aim 1 against human sequences to identify homologous genes that may locate in human mitochondria. We will screen each gene discovered for possible mutations in a collection of more than cell lines obtained from patients with phenotypes known or assumed to be associated with mutations in mitochondrial proteins. These results together with data obtained with human expression arrays shall help identify new disease related genes and pathways. For these proteomic studies, methods have been developed to successfully isolate highly purified yeast mitochondria membranes, as measured by antibody binding assays as well as 2-D gel electrophoresis. Research is underway to achieve the same quality for isolation of mitochondria from human cell lines.
Project Details
Project type
Exploratory Research
Start Date
2002-02-28
End Date
2003-09-12
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Prokisch H, CM Scharfe, DG Camp, W Xiao, L David, C Andreoli, ME Monroe, RJ Moore, MA Gritsenko, C Kozany, KK Hixson, HM Mottaz, H Zischka, M Ueffing, ZS Herman, RW Davis, T Meitinger, P Oefner, RD Smith, and LM Steinmetz. 2004. "Integrative Analysis of the Mitochondrial Proteome in Yeast." PloS Biology 2(6):0795-0804.