Genetic Analysis of Resistance and Sensitivity to 2-Deoxyglucose in Saccharomyces cerevisiae

Aerobic glycolysis is a metabolic pathway utilized by human cancer cells and also by yeast cells when they ferment glucose to ethanol. Both cancer cells and yeast cells are inhibited by the presence of low concentrations of 2-deoxyglucose (2DG). Genetic screens in yeast used resistance to 2-deoxyglucose to identify a small set of genes that function in regulating glucose metabolism. A recent high throughput screen for 2-deoxyglucose resistance identified a much larger set of seemingly unrelated genes. Here, we demonstrate that these newly identified genes do not in fact confer significant resistance to 2-deoxyglucose. Further, we show that the relative toxicity of 2-deoxyglucose is carbon source dependent, as is the resistance conferred by gene deletions. Snf1 kinase, the AMP-activated protein kinase of yeast, is required for 2-deoxyglucose resistance in cells growing on glucose. Mutations in the SNF1 gene that reduce kinase activity render cells hypersensitive to 2-deoxyglucose, while an activating mutation in SNF1 confers 2-deoxyglucose resistance. Snf1 kinase activated by 2-deoxyglucose does not phosphorylate the Mig1 protein, a known Snf1 substrate during glucose limitation. Thus, different stimuli elicit distinct responses from the Snf1 kinase.     

FIGfams: yet another set of protein families

We present FIGfams, a new collection of over 100 000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950 000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license. These can be downloaded at ftp://ftp.theseed.org/FIGfams/. The web site for FIGfams is http://www.theseed.org/wiki/FIGfams/     

Remodeling of the Rad51 DNA Strand-Exchange Protein by the Srs2 Helicase

Homologous recombination is associated with the dynamic assembly and disassembly of DNA–protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.     

Genetic Networks Inducing Invasive Growth in Saccharomyces cerevisiae Identified Through Systematic Genome-Wide Overexpression

The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This growth transition has been studied extensively as a model signaling system with similarity to processes of hyphal development that are linked with virulence in related fungal pathogens. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. Here, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. This cohort includes 79 genes lacking previous phenotypic characterization. Pathway enrichment analysis of the gene set identifies networks mediating mitogen-activated protein kinase (MAPK) signaling and cell cycle progression. In particular, overexpression screening suggests that nuclear export of the osmoresponsive MAPK Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. Through epistasis and deletion studies, we also identified genetic relationships with the G2 cyclin Clb2p and phenotypes in filamentation induced by S-phase arrest. In sum, this work presents a unique and informative resource toward understanding the breadth of genes and pathways that collectively constitute the molecular basis of filamentation.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

PathVisio 3: An Extendable Pathway Analysis Toolbox

PathVisio is a commonly used pathway editor, visualization and analysis software. Biological pathways have been used by biologists for many years to describe the detailed steps in biological processes. Those powerful, visual representations help researchers to better understand, share and discuss knowledge. Since the first publication of PathVisio in 2008, the original paper was cited more than 170 times and PathVisio was used in many different biological studies. As an online editor PathVisio is also integrated in the community curated pathway database WikiPathways.Here we present the third version of PathVisio with the newest additions and improvements of the application. The core features of PathVisio are pathway drawing, advanced data visualization and pathway statistics. Additionally, PathVisio 3 introduces a new powerful extension systems that allows other developers to contribute additional functionality in form of plugins without changing the core application.PathVisio can be downloaded from http://www.pathvisio.org and in 2014 PathVisio 3 has been downloaded over 5,500 times. There are already more than 15 plugins available in the central plugin repository. PathVisio is a freely available, open-source tool published under the Apache 2.0 license (http://www.apache.org/licenses/LICENSE-2.0). It is implemented in Java and thus runs on all major operating systems. The code repository is available at http://svn.bigcat.unimaas.nl/pathvisio. The support mailing list for users is available on https://groups.google.com/forum/#!forum/wikipathways-discuss and for developers on https://groups.google.com/forum/#!forum/wikipathways-devel.

This is a PLOS Computational Biology software article.

     

The Jaw of the Worm: GTPase-activating Protein EAT-17 Regulates Grinder Formation in Caenorhabditis elegans

Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.     

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs.     

Translational Control of the Oogenic Program by Components of OMA Ribonucleoprotein Particles in Caenorhabditis elegans

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3′-untranslated regions (3′-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.     

TRIP Database 2.0: A Manually Curated Information Hub for Accessing TRP Channel Interaction Network

Transient receptor potential (TRP) channels are a family of Ca²⁺-permeable cation channels that play a crucial role in biological and disease processes. To advance TRP channel research, we previously created the TRIP (TRansient receptor potential channel-Interacting Protein) Database, a manually curated database that compiles scattered information on TRP channel protein-protein interactions (PPIs). However, the database needs to be improved for information accessibility and data utilization. Here, we present the TRIP Database 2.0 (http://www.trpchannel.org) in which many helpful, user-friendly web interfaces have been developed to facilitate knowledge acquisition and inspire new approaches to studying TRP channel functions: 1) the PPI information found in the supplementary data of referred articles was curated; 2) the PPI summary matrix enables users to intuitively grasp overall PPI information; 3) the search capability has been expanded to retrieve information from ‘PubMed’ and ‘PIE the search’ (a specialized search engine for PPI-related articles); and 4) the PPI data are available as sif files for network visualization and analysis using ‘Cytoscape’. Therefore, our TRIP Database 2.0 is an information hub that works toward advancing data-driven TRP channel research.     

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication.     

State Transitions in the TORC1 Signaling Pathway and Information Processing in Saccharomyces cerevisiae

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases—Gtr1, Gtr2, and Rho1—bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.     

Suppression of Chromosome Healing and Anticheckpoint Pathways in Yeast Postsenescence Survivors

Telomere repeat-like sequences at DNA double-strand breaks (DSBs) inhibit DNA damage signaling and serve as seeds to convert DSBs to new telomeres in mutagenic chromosome healing pathways. We find here that the response to seed-containing DSBs differs fundamentally between budding yeast (Saccharomyces cerevisiae) cells that maintain their telomeres via telomerase and so-called postsenescence survivors that use recombination-based alternative lengthening of telomere (ALT) mechanisms. Whereas telomere seeds are efficiently elongated by telomerase, they remain remarkably stable without de novo telomerization or extensive end resection in telomerase-deficient (est2Δ, tlc1Δ) postsenescence survivors. This telomere seed hyper-stability in ALT cells is associated with, but not caused by, prolonged DNA damage checkpoint activity (RAD9, RAD53) compared to telomerase-positive cells or presenescent telomerase-negative cells. The results indicate that both chromosome healing and anticheckpoint activity of telomere seeds are suppressed in yeast models of ALT pathways.     

Complete genome sequence of the marine methyl-halide oxidizing Leisingera methylohalidivorans type strain (DSM 14336T), a representative of the Roseobacter clade

Leisingera methylohalidivorans Schaefer et al. 2002 emend. Vandecandelaere et al. 2008 is the type species of the genus Leisingera. The genus belongs to the Roseobacter clade (Rhodobacteraceae, Alphaproteobacteria), a widely distributed lineage in marine environments. Leisingera and particularly L. methylohalidivorans strain MB2^T is of special interest due to its methylotrophy. Here we describe the complete genome sequence and annotation of this bacterium together with previously unreported aspects of its phenotype. The 4,650,996 bp long genome with its 4,515 protein-coding and 81 RNA genes consists of three replicons, a single chromosome and two extrachromosomal elements with sizes of 221 kb and 285 kb.     

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.     

Sequence Similarity Network Reveals Common Ancestry of Multidomain Proteins

We address the problem of homology identification in complex multidomain families with varied domain architectures. The challenge is to distinguish sequence pairs that share common ancestry from pairs that share an inserted domain but are otherwise unrelated. This distinction is essential for accuracy in gene annotation, function prediction, and comparative genomics. There are two major obstacles to multidomain homology identification: lack of a formal definition and lack of curated benchmarks for evaluating the performance of new methods. We offer preliminary solutions to both problems: 1) an extension of the traditional model of homology to include domain insertions; and 2) a manually curated benchmark of well-studied families in mouse and human. We further present Neighborhood Correlation, a novel method that exploits the local structure of the sequence similarity network to identify homologs with great accuracy based on the observation that gene duplication and domain shuffling leave distinct patterns in the sequence similarity network. In a rigorous, empirical comparison using our curated data, Neighborhood Correlation outperforms sequence similarity, alignment length, and domain architecture comparison. Neighborhood Correlation is well suited for automated, genome-scale analyses. It is easy to compute, does not require explicit knowledge of domain architecture, and classifies both single and multidomain homologs with high accuracy. Homolog predictions obtained with our method, as well as our manually curated benchmark and a web-based visualization tool for exploratory analysis of the network neighborhood structure, are available at http://www.neighborhoodcorrelation.org. Our work represents a departure from the prevailing view that the concept of homology cannot be applied to genes that have undergone domain shuffling. In contrast to current approaches that either focus on the homology of individual domains or consider only families with identical domain architectures, we show that homology can be rationally defined for multidomain families with diverse architectures by considering the genomic context of the genes that encode them. Our study demonstrates the utility of mining network structure for evolutionary information, suggesting this is a fertile approach for investigating evolutionary processes in the post-genomic era.