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1.  Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining 
Molecular and Cellular Biology  2015;35(14):2448-2463.
The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.
doi:10.1128/MCB.00007-15
PMCID: PMC4475915  PMID: 25963654
2.  Mitochondrial Targets for Pharmacological Intervention in Human Disease 
Journal of Proteome Research  2014;14(1):5-21.
Over the past several years, mitochondrial dysfunction has been linked to an increasing number of human illnesses, making mitochondrial proteins (MPs) an ever more appealing target for therapeutic intervention. With 20% of the mitochondrial proteome (312 of an estimated 1500 MPs) having known interactions with small molecules, MPs appear to be highly targetable. Yet, despite these targeted proteins functioning in a range of biological processes (including induction of apoptosis, calcium homeostasis, and metabolism), very few of the compounds targeting MPs find clinical use. Recent work has greatly expanded the number of proteins known to localize to the mitochondria and has generated a considerable increase in MP 3D structures available in public databases, allowing experimental screening and in silico prediction of mitochondrial drug targets on an unprecedented scale. Here, we summarize the current literature on clinically active drugs that target MPs, with a focus on how existing drug targets are distributed across biochemical pathways and organelle substructures. Also, we examine current strategies for mitochondrial drug discovery, focusing on genetic, proteomic, and chemogenomic assays, and relevant model systems. As cell models and screening techniques improve, MPs appear poised to emerge as relevant targets for a wide range of complex human diseases, an eventuality that can be expedited through systematic analysis of MP function.
doi:10.1021/pr500813f
PMCID: PMC4286170  PMID: 25367773
Drug−protein interactions; human disease; mitochondria; model system; network; pharmacological target; protein complex; pathways; small molecules; systems biology
3.  Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes 
Molecular Biology of the Cell  2015;26(6):1119-1128.
The retromer complex regulates vesicle transport at endosomes. Different members of the VPS9 domain–containing Rab5-family guanine nucleotide exchange factors interact with the yeast retromer complex and mediate its endosomal localization.
The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.
doi:10.1091/mbc.E14-08-1281
PMCID: PMC4357511  PMID: 25609093
4.  Mapping the functional yeast ABC transporter interactome 
Nature chemical biology  2013;9(9):565-572.
ABC transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used Membrane Yeast Two-Hybrid (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated interactome. We show that ABC transporters physically associate with proteins involved in a surprisingly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.
doi:10.1038/nchembio.1293
PMCID: PMC3835492  PMID: 23831759
5.  Phosphatase Complex Pph3/Psy2 Is Involved in Regulation of Efficient Non-Homologous End-Joining Pathway in the Yeast Saccharomyces cerevisiae 
PLoS ONE  2014;9(1):e87248.
One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.
doi:10.1371/journal.pone.0087248
PMCID: PMC3909046  PMID: 24498054
6.  Short Co-occurring Polypeptide Regions Can Predict Global Protein Interaction Maps 
Scientific Reports  2012;2:239.
A goal of the post-genomics era has been to elucidate a detailed global map of protein-protein interactions (PPIs) within a cell. Here, we show that the presence of co-occurring short polypeptide sequences between interacting protein partners appears to be conserved across different organisms. We present an algorithm to automatically generate PPI prediction method parameters for various organisms and illustrate that global PPIs can be predicted from previously reported PPIs within the same or a different organism using protein primary sequences. The PPI prediction code is further accelerated through the use of parallel multi-core programming, which improves its usability for large scale or proteome-wide PPI prediction. We predict and analyze hundreds of novel human PPIs, experimentally confirm protein functions and importantly predict the first genome-wide PPI maps for S. pombe (∼9,000 PPIs) and C. elegans (∼37,500 PPIs).
doi:10.1038/srep00239
PMCID: PMC3269044  PMID: 22355752
7.  Ribosome-Dependent ATPase Interacts with Conserved Membrane Protein in Escherichia coli to Modulate Protein Synthesis and Oxidative Phosphorylation 
PLoS ONE  2011;6(4):e18510.
Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.
doi:10.1371/journal.pone.0018510
PMCID: PMC3083400  PMID: 21556145
8.  Chemical-genetic profile analysis of five inhibitory compounds in yeast 
BMC Chemical Biology  2010;10:6.
Background
Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s).
Results
Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays.
Conclusion
Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.
doi:10.1186/1472-6769-10-6
PMCID: PMC2925817  PMID: 20691087
9.  Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis 
BMC Genomics  2008;9:583.
Background
Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis.
Results
As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis.
Conclusion
We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).
doi:10.1186/1471-2164-9-583
PMCID: PMC2613417  PMID: 19055778
10.  Colony size measurement of the yeast gene deletion strains for functional genomics 
BMC Bioinformatics  2007;8:117.
Background
Numerous functional genomics approaches have been developed to study the model organism yeast, Saccharomyces cerevisiae, with the aim of systematically understanding the biology of the cell. Some of these techniques are based on yeast growth differences under different conditions, such as those generated by gene mutations, chemicals or both. Manual inspection of the yeast colonies that are grown under different conditions is often used as a method to detect such growth differences.
Results
Here, we developed a computerized image analysis system called Growth Detector (GD), to automatically acquire quantitative and comparative information for yeast colony growth. GD offers great convenience and accuracy over the currently used manual growth measurement method. It distinguishes true yeast colonies in a digital image and provides an accurate coordinate oriented map of the colony areas. Some post-processing calculations are also conducted. Using GD, we successfully detected a genetic linkage between the molecular activity of the plant-derived antifungal compound berberine and gene expression components, among other cellular processes. A novel association for the yeast mek1 gene with DNA damage repair was also identified by GD and confirmed by a plasmid repair assay. The results demonstrate the usefulness of GD for yeast functional genomics research.
Conclusion
GD offers significant improvement over the manual inspection method to detect relative yeast colony size differences. The speed and accuracy associated with GD makes it an ideal choice for large-scale functional genomics investigations.
doi:10.1186/1471-2105-8-117
PMCID: PMC1854909  PMID: 17408490

Results 1-10 (10)