The yeast ubiquitin ligase SCF(Met30): connecting environmental and intracellular conditions to cell division

Ubiquitination regulates a host of cellular processes and is well known for its role in progression through the cell division cycle. In budding yeast, cadmium and arsenic stress, the availability of sulfur containing amino acids, and the intracellular concentration of S-adenosylmethionine are linked to cell cycle regulation through the ubiquitin ligase SCF(Met30). Regulation is achieved by ubiquitination of the transcription factor Met4. Met4 activity is controlled by a regulatory K48-linked ubiquitin chain that is synthesized by Cdc34/SCF(Met30). A ubiquitin-interacting-motif (UIM) present in Met4 prevents degradation of ubiquitinated Met4 allowing the ubiquitin chain to function as a reversible switch of Met4 activity. Here we discuss mechanisms of Met4 and SCF(Met30 )regulation in response to intracellular and environmental conditions, and describe the integration of these signals with cell cycle control

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) tocentromeres is essential for faithful chromosome segregation. Mislocalization of CENP-Aleads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression andmislocalization of CENP-A has been observed in many cancers and this correlates withincreased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels andlocalization under physiological conditions have not been defined. In this study we used agenome-wide genetic screen to identify essential genes required for Cse4 homeostasis toprevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, Fbox(SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 andCdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent itsmislocalization for faithful chromosome segregation under physiological conditions. Theinteraction of Met30 with Cdc4 is independent of the D domain, which is essential for theirhomodimerization and ubiquitination of other substrates. The requirement for both Cdc4and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 andMet30 has not previously been described. Met30 is necessary for the interaction betweenCdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization ofCse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalizationto defects in kinetochore structure and show that SCF-mediated proteolysis ofPLOS Genetics Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromericregions, thus ensuring faithful chromosome segregation. In summary, we have identifiedessential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysisof Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.Fil: Au, Wei-Chun. National Institutes of Health; Estados UnidosFil: Zhang, Tianyi. National Institutes of Health; Estados UnidosFil: Mishra, Prashant K.. National Institutes of Health; Estados UnidosFil: Eisenstatt, Jessica R.. National Institutes of Health; Estados UnidosFil: Walker, Robert L.. National Institutes of Health; Estados UnidosFil: Ocampo, Josefina. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Dawson, Anthony. National Institutes of Health; Estados UnidosFil: Warren, Jack. National Institutes of Health; Estados UnidosFil: Costanzo, Michael. University of Toronto; CanadáFil: Baryshnikova, Anastasia. California Life Company; Estados UnidosFil: Flick, Karin. University of California; Estados UnidosFil: Clark, David J.. National Institutes of Health; Estados UnidosFil: Meltzer, Paul S.. National Institutes of Health; Estados UnidosFil: Baker, Richard E.. University of Massachussets; Estados UnidosFil: Myers, Chad. University of Minnesota; Estados UnidosFil: Boone, Charles. University of Toronto; CanadáFil: Kaiser, Peter. University of California; Estados UnidosFil: Basrai, Munira A.. National Institutes of Health; Estados Unido

Community acceptability of use of rapid diagnostic tests for malaria by community health workers in Uganda

<p>Abstract</p> <p>Background</p> <p>Many malarious countries plan to introduce artemisinin combination therapy (ACT) at community level using community health workers (CHWs) for treatment of uncomplicated malaria. Use of ACT with reliance on presumptive diagnosis may lead to excessive use, increased costs and rise of drug resistance. Use of rapid diagnostic tests (RDTs) could address these challenges but only if the communities will accept their use by CHWs. This study assessed community acceptability of the use of RDTs by Ugandan CHWs, locally referred to as community medicine distributors (CMDs).</p> <p>Methods</p> <p>The study was conducted in Iganga district using 10 focus group discussions (FGDs) with CMDs and caregivers of children under five years, and 10 key informant interviews (KIIs) with health workers and community leaders. Pre-designed FGD and KII guides were used to collect data. Manifest content analysis was used to explore issues of trust and confidence in CMDs, stigma associated with drawing blood from children, community willingness for CMDs to use RDTs, and challenges anticipated to be faced by the CMDs.</p> <p>Results</p> <p>CMDs are trusted by their communities because of their commitment to voluntary service, access, and the perceived effectiveness of anti-malarial drugs they provide. Some community members expressed fear that the blood collected could be used for HIV testing, the procedure could infect children with HIV, and the blood samples could be used for witchcraft. Education level of CMDs is important in their acceptability by the community, who welcome the use of RDTs given that the CMDs are trained and supported. Anticipated challenges for CMDs included transport for patient follow-up and picking supplies, adults demanding to be tested, and caregivers insisting their children be treated instead of being referred.</p> <p>Conclusion</p> <p>Use of RDTs by CMDs is likely to be acceptable by community members given that CMDs are properly trained, and receive regular technical supervision and logistical support. A well-designed behaviour change communication strategy is needed to address the anticipated programmatic challenges as well as community fears and stigma about drawing blood. Level of formal education may have to be a criterion for CMD selection into programmes deploying RDTs.</p

Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)^(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF^(Met30) in vivo and in vitro, but not the closely related SCF^(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes

Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2–Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UASINO-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UASINO-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways

Multiple Pathways for Suppression of Mutants Affecting G(1)-Specific Transcription in Saccharomyces cerevisiae

In the budding yeast, Saccharomyces cerevisiae, control of cell proliferation is exerted primarily during G(1) phase. The G(1)-specific transcription of several hundred genes, many with roles in early cell cycle events, requires the transcription factors SBF and MBF, each composed of Swi6 and a DNA-binding protein, Swi4 or Mbp1, respectively. Binding of these factors to promoters is essential but insufficient for robust transcription. Timely transcriptional activation requires Cln3/CDK activity. To identify potential targets for Cln3/CDK, we identified multicopy suppressors of the temperature sensitivity of new conditional alleles of SWI6. A bck2Δ background was used to render SWI6 essential. Seven multicopy suppressors of bck2Δ swi6-ts mutants were identified. Three genes, SWI4, RME1, and CLN2, were identified previously in related screens and shown to activate G(1)-specific expression of genes independent of CLN3 and SWI6. The other four genes, FBA1, RPL40a/UBI1, GIN4, and PAB1, act via apparently unrelated pathways downstream of SBF and MBF. Each depends upon CLN2, but not CLN1, for its suppressing activity. Together with additional characterization these findings indicate that multiple independent pathways are sufficient for proliferation in the absence of G(1)-specific transcriptional activators

The F-Box Protein Met30 Is Required for Multiple Steps in the Budding Yeast Cell Cycle

The Saccharomyces cerevisiae ubiquitin ligase SCF(Met30) is essential for cell cycle progression. To identify and characterize SCF(Met30)-dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G(1)/S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G(1)-specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G(1) arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G(1)-specific transcription, initiation of DNA replication, and progression through M phase

Ubiquitin and transcription: The SCFMet30/Met4 pathway, a (protein-) complex issue

Ubiquitylation has emerged as an omnipresent factor at all levels of transcriptional regulation. A recent study that describes the yeast transcriptional activator Met4 as a functional component of the very same ubiquitin ligase that regulates its own activity highlights the close relation between transcription and the ubiquitin proteasome system