Yeast Deletion Project and Proteomics of Mitochondria Database

 
Description

The YDPM database serves to support the Yeast Deletion and the Mitochondrial Proteomics Project. The database is hyperlinked with other public databases. The project aims to increase the understanding of mitochondrial function and biogenesis in the context of the cell.

In the Deletion Project, strains from the deletion collection were monitored under 9 different media conditions selected for the study of mitochondrial function. 5791 heterozygous diploid and 4706 homozygous diploid deletion strains were monitored in parallel using molecular barcodes on fermentable (YPD, YPDGE) and non-fermentable substrates (YPG, YPE, YPL). The YDPM database contains both the raw data and growth rates calculated for each strain in each media condition. Strains can be searched by ORF or Gene name to access growth measurements and data plots for each strain.

In the Proteomics Project, mitochondria were purified from yeast cells cultured under 4 different growth conditions, including fermentable (glucose) and non-fermentable (lactate) substrates for both natural and synthetic culture media (YP* and SC*). Density gradient and free flow electrophoresis were used independently to purify whole mitochondrial organelles. Preparations were separated into mitochondrial membrane and matrix fractions and analyzed both by LC-MS/MS and LC-FTICR (Fourier Transformed Ion Cyclotron Resonance) mass spectrometry. The YDPM database contains the mass and time tag values detected by mass spectrometry which were used to define the corresponding protein through a yeast proteome database search.

The Expression Project contains genome-wide measurements and analysis of mRNA expression values from Log phase cultures grown under the same media conditions as used for the proteomics study (YPD, SCD, YPL, SCL). Total RNA was isolated using a hot phenol glass-bead lysis protocol and polyA+ mRNA was purified using Qiagen oligotex kit. After reverse transcrition to single stranded cDNA, products were fragmented to 50bp using Dnase digestion, biotin end labeled and hybridized to Affymetrix S98 high-density arrays as described in the Affymetrix user handbook (Affymetrix, Santa Clara). Hybridizations were normalized and duplicate samples integrated to arrive at an estimate of absolute transcript abundance using the dChip computational package (Harvard University). For genes with multiple probe sets on the array only the probe set with the highest signal was used.


Specific Information on the links:

The Analysis is subdivided for the 3 different projects for the study of mitochondrial function and biogenesis.

In the Deletion Project, the homozygous diploid strains were grouped into three classes: class I lists genes without significant growth differences between conditions; class II has genes with a fermentable growth defect; class III genes with a non-fermentable growth deficiency. Each list is interlinked with the database. In an additional analysis class IIIa and class IIIb were combined (different degrees of non-fermentable growth defects) and narrowed to 466 genes by removing spurious ORFs. The 466 genes were divided into 201 previously known mitochondrial genes (localization or function); 161 with unknown localization; 104 localized outside mitochondria. Important: These numbers reflect the status on the time of the publication. To date, of the 466 genes detected with a quantitative deletion phenotype under non-fermentable growth conditions, 225 are confirmed with a mitochondrial localization.

In the Proteomics Project, mass and time tags for 2086 different proteins were detected with 17% (361) known to be localized to mitochondria (about two times the level in the genome). For proteins identified with 2 tags or more, the percentage is 54% (256 mitochondrial proteins in total 546 candidates). Of the 546 candidate proteins, 203 have a known localization outside the mitochondrial organelle (nucleus, cytoplasm, ER), and 87 are of unknown localization. The list of 546 candidate proteins was divided into three classes that define the confidence of identification. Classes are High (209 proteins), Medium (228) or Low (109). Similar numbers of proteins were identified from membrane and matrix fractions, indicating that, compared to the 477 reference set, we observed no bias in representation of proteins associated with membrane and soluble fractions.

The Expression, study identified 870 genes with at least 1.2-fold expression level differences between fermentable and non-fermentable growth conditions. 100 proteins have a known mitochondrial localization, while 513 are located outside the organelle. 257 of the measured proteins are unclassified without a known subcellular localization.

Under Homology yeast genes are listed that are similar to proteins in Rickettsia prowazeckii and bacteria in general.

Under Prediction yeast genes are listed according to whether they are known mitochondria-related proteins (MitoP and the new Mitop2 database) or have a high probability of containing a mitochondrial import sequence (Mitoprot).

Under External data are functional and phenotypic classifications acquired from public databases, mostly from CYGD.

All data are available for download from the deletion project for homozygous and heterozygous pools (two repeat experiments), the twenty-eight datasets generated in the proteomics project, and the zipped Affymetrix CEL files from the mRNA expression study (two repeat experiments).


Database citation: Data provided in YDPM were generated and further used for analysis in the following studies:

Systematic screen for human disease genes in yeast. Steinmetz LM, Scharfe C, Deutschbauer AM, Mokranjac D, Herman ZS, Jones T, Chu AM, Giaever G, Prokisch H, Oefner PJ, Davis RW. Nat Genet. 2002 Aug;31(4):400-4.

Evolutionary rate in the protein interaction network. Fraser HB, Hirsh AE, Steinmetz LM, Scharfe C, Feldman MW. Science. 2002 Apr 26;296(5568):750-2.

Role of duplicate genes in genetic robustness against null mutations. Gu Z, Steinmetz LM, Gu X, Scharfe C, Davis RW, Li WH. Nature. 2003 Jan 2;421(6918):63-6.

Dosage sensitivity and the evolution of gene families in yeast. Papp B, Pal C, Hurst LD. Nature. 2003 Jul 10;424(6945):194-7.

Integrative analysis of the mitochondrial proteome in yeast. Prokisch H, Scharfe C, Camp DG 2nd, Xiao W, David L, Andreoli C, Monroe ME, Moore RJ, Gritsenko MA, Kozany C, Hixson KK, Mottaz HM, Zischka H, Ueffing M, Herman ZS, Davis RW, Meitinger T, Oefner PJ, Smith RD, Steinmetz LM. PLoS Biol. 2004 Jun;2(6):e160. Epub 2004 Jun 15.


Web site © 2003 Stanford Genome Technology Center

In addition to original data, this Web site and its associated links contain information gleened from public data. While the public data has been faithfully downloaded into the YDPM database, no representations are made regarding the accuracy of the public data.

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