Reciprocal Hemizygosity Analysis

<p>The vertical lines represent the chromosome XIV QTL, and the boxes on the line represent individual genes in the Htg QTL. In RHA, for each Htg QTG <i>(RHO2, MKT1,</i> and <i>END3),</i> one allele of each QTG (or putative QTG) is deleted in a hybrid strain resulting in a pair of deletion heterozygotes (hemizygotes) that have one intact QTG allele from each parent. RHA competitions between the strains in a pair are done to determine the contribution of each Htg allele to the Htg phenotype.</p

Properties of Yeast Orthologs to Human Disease Genes within the Mitochondrial System

<div><p>(A) Five modules enriched in human disease gene orthologs. Node color identifies human orthologs to yeast genes with and without associated Mendelian diseases (OMIM database). Proteins that belong to physical complexes are shown by overlapping nodes or in some cases are connected by solid lines; functional associations are shown by dotted lines. Disease genes within the same functional module had a tendency to have similar clinical phenotypes: glutaricaciduria II (NAD metabolism/tricarboxylic acid cycle), glycine encephalopathy (folate and glycine metabolism), mainly susceptibility to hereditary pheochromocytoma and paraganglioma (RCC-II), and variants of inherited disease of porphyrin metabolism (heme biosynthesis). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020170#pgen-0020170-st006" target="_blank">Table S6</a> contains descriptions for each disease gene.</p><p>(B) Conservation of disease genes to proteobacteria. Venn diagram of the overlap of yeast genes having human orthologs, proteobacterial orthologs, and human disease gene orthologs. Of the proteins with a disease ortholog, 31% (31/99) have a proteobacterial ortholog—whereas only 18% (100/565) of all human orthologs have a proteobacterial ortholog (hypergeometric test, <i>p</i> < 10⁻⁴).</p><p>(C) Fitness distribution of yeast orthologs of disease genes. Empirical cumulative distributions of NF fitness scores are shown for all human disease gene orthologs compared to the remaining human orthologs. NF fitness represents the growth and survival of single gene deletion mutants under respiratory conditions (NF carbon source): higher fitness represents a milder growth defect. As a group, disease orthologs have higher fitness (i.e., milder mutant phenotypes) (<i>t</i>-test, <i>p</i> < 0.01).</p></div

Functional Network of the Mitochondrial System

<div><p>(A) Full network containing 9,780 association lines connecting 876 protein nodes. Lines are shaded by the degree of STRING confidence in the association. Nodes are colored according to the following: known mitochondrial-localized proteins (reference set) correctly predicted by the linear classifier, green; known mitochondrial-localized proteins not captured by the linear classifier, light green; predicted proteins not annotated as mitochondrial-localized (mitochondrial candidates), orange; proteins predicted as additional interactors by the network (interactor candidates), blue; mitochondrial candidates recently annotated as mitochondrial-localized (MitoP2 database) or verified by mitochondrial import assay, red.</p><p>(B) Module map of 46 modules with five or more proteins. Modules were named and localized based on GO terms, with the following abbreviations: asm, assembly; biogen, biogenesis; cyt, cytoplasmic; dehy, dehydrogenase; met, metabolism; mito, mitochondrial; org, organization; proces, processing; syn, synthesis. The localization of modules in three different compartments—nucleus, mitochondria, and cytoplasm—is indicated by sectors of different colors. When the module contains a mixture of proteins with different localization it is annotated as shared between the different compartments. Module shared between mitochondria and nucleus or mitochondria and cytoplasm belong to green and yellow sectors, respectively. Cytoplasm refers to all of the contents of a cell excluding mitochondrion and nucleus but including the plasma membrane and other sub-cellular structures. The identity of all proteins and their functional links can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020170#pgen-0020170-sg004" target="_blank">Figure S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020170#pgen-0020170-sg005" target="_blank">Figure S5</a> for (A and B), respectively, where the standard gene names are shown within the nodes and are hyperlinked to STRING.</p></div

Verification of Predicted Mitochondrial Candidates by Mitochondrial Import

<p>Samples were derived by incubating radiolabeled proteins with isolated mitochondria in the presence or absence of a membrane potential and of proteinase K. Cases where import was accompanied by removal of the signal peptide are marked as ‘‘SP-processing'' (+). Su9(1–69)DHFR and AAC serve as positive controls for a processed matrix protein and a non-processed inner membrane protein, respectively. The score reflects the likelihood of mitochondrial localization for tested candidates as predicted by the linear classifier. MP, membrane potential; PK, proteinase K; SP, signal peptide.</p

Origin and Conservation of the Mitochondrial System

<p>Orthology was used to identify functional processes in yeast that originate from bacteria and are conserved to humans. Modules of five or more proteins are shown as single nodes at the circumference of the circle and the degree of evidence connecting modules is shown by lines in different gray tone. The connection between modules is calculated as the average of the STRING interaction scores for all protein-pairs. Modules are distributed according to their localization. Color in the inner rings reflects for each module the percentage of proteins that have proteobacterial orthology (red) or human orthology (blue). The number of proteobacterial orthologs is also indicated.</p

Summary of the Integrated Approach

<p>Summary of the Integrated Approach</p

Enrichment for Mitochondrial Proteins by MS

<div><p>(A) shows the 546 proteins (in rows) identified from 28 datasets (columns). The proteins are sorted in decreasing order down rows by the number of experiments in which peptide tags were identified by MS and binned into three classes of detection frequency. The number at the bottom of each class indicates the total number of proteins in the class. Proteins that are part of the reference set, and thus are previously known mitochondrial proteins (M), are marked to the left. The experiments are divided according to fermentable (F) and nonfermentable (NF) mitochondrial preparations.</p> <p>(B) Proportions of proteins identified in membrane and matrix fractions. Whether a protein was detected predominantly in either the membrane or matrix fraction, or equal in both fractions, was determined based on where it was detected with an average higher tag number. Shown are the proportions for all 546 proteins, for known matrix proteins (i.e., matrix and intermembrane space, <i>n</i> = 109), for known membrane proteins (i.e., inner and outer membrane, <i>n</i> = 101), and for detected proteins not previously known to be mitochondrial (<i>n</i> = 290).</p> <p>(C) Distribution of proteins identified under fermentable and nonfermentable conditions by proteomics, and overlap with previously known mitochondrial proteins. Total numbers are given in parentheses.</p> <p>(D) Breakdown by localization of the 546 proteins identified. For mitochondrial localization the reference set was chosen; for localization outside mitochondria the GFP fusion protein data were used (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#pbio-0020160-Huh1" target="_blank">Huh et al. 2003</a>). The inner circle represents the distribution for all proteins in yeast.</p> <p>(E) Distribution of median mRNA expression under fermentable and nonfermentable conditions, protein abundance under fermentable conditions (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#pbio-0020160-Ghaemmaghami1" target="_blank">Ghaemmaghami et al. 2003</a>), and protein length across bins of confidence of identification (maximum number of tags identified in any of the 28 datasets). The bars indicate fold differences from the median for the known mitochondrial proteins that were not detected by MS (“M not det.”).</p></div

Verification of Prediction in Selected Mitochondrial Protein Complexes

<p>The assignment of complexes to mitochondrial compartments is based on known localizations of the protein subunits. Complexes are shown as clusters of circles, where each circle represents one protein. Red denotes a protein that was detected under fermentable and green under nonfermentable growth conditions by our proteomic dataset; white indicates proteins that were not detected. The numbers indicate the MitoP2 predictive score. For proteins without a number, no predictive score was assigned by the integrative analysis. Ac, acetyl; CoA, coenzyme A; α-KG, α-ketoglutarate; GDC, glycine decarboxylase; NDH, NADH-oxidoreductase; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; RCC, respiratory chain complex; TIM, transport across inner membrane; TOM, transport across outer membrane; MOM and MIM, mitochondrial outer and inner membrane, respectively. A list of the genes for the plotted complexes is available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#st004" target="_blank">Table S4</a>.</p

Verification of Proteomic Candidates by Mitochondrial Import

<p>Samples were incubated in the presence or absence of a membrane potential (MP) and of proteinase K (PK). Cases where import was accompanied by removal of the signal peptide (SP) are marked as “SP-processing” (+). Su9(1–69)DHFR and AAC serve as positive controls for a processed matrix protein and a nonprocessed inner membrane protein, respectively. The bar graphs indicate if a protein was more likely to be found in either the membrane or the matrix fractions of our proteomic data. The height of the bar corresponds to the number of samples in which a protein was identified with higher tag number—in the mitochondrial membrane or mitochondrial matrix fractions, respectively.</p

Evaluation of Proteomic Data for Protein Abundance and Mitochondrial Localization

<div><p>(A) Coverage of known mitochondrial proteins (Mref) by two MS proteome studies (this study and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#pbio-0020160-Sickmann1" target="_blank">Sickmann et al. [2003]</a>). We evaluated the 340 proteins of the mitochondrial reference set for which protein abundance data existed (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#pbio-0020160-Ghaemmaghami1" target="_blank">Ghaemmaghami et al. 2003</a>). The x-axis represents the median protein abundance of ten consecutive, equally sized bins of proteins.</p> <p>(B) Distribution and overlap of proteins identified by the two MS studies and known mitochondrial proteins. The total number of entries for each dataset is indicated in parentheses outside each circle. The number inside each circle indicates the number of proteins in each of the categories. In addition, the percentage of proteins that were localized to mitochondria by GFP tagging (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020160#pbio-0020160-Huh1" target="_blank">Huh et al. 2003</a>) is given in parentheses for each category.</p></div