A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.     

A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.     

A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes.     

A genomic screen for yeast mutants defective in mitophagy

Mitochondria autophagy (mitophagy) is the process of selective degradation of mitochondria that has an important role in mitochondrial quality control. To gain insight into the molecular mechanism of mitophagy, we screened a yeast knockout library for strains that are defective in mitophagy. We found 32 strains that showed a complete or partial block of mitophagy. One of the genes identified, YLR356W, is required for mitophagy, but not for macroautophagy or other types of selective autophagy. The deletion of YLR356W partially inhibits mitophagy during starvation, whereas there is almost complete inhibition at post-log phase. Accordingly, we hypothesize that Ylr356w is required to detect or present aged or dysfunctional mitochondria when cells reach the post-log phase.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.     

A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae.

Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation. Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates. Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years. We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene. This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome. Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation. The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.     

A yeast two hybrid screen identifies SPATA4 as a TRAPP interactor

The TRAPP vesicle-tethering complex consists of more than 10 distinct polypeptides and is involved in protein transport. Using the C2 subunit as bait we identified SPATA4, a spermatocyte-specific protein of unknown function, as an interacting partner in a yeast two hybrid screen. Further studies indicate SPATA4 interacts with the C2 portion of the TRAPP complex. SPATA4 fractionates with both cytosolic and nuclear fractions suggesting it may have several distinct functions. SPATA4 is one of only three human proteins that contain a DUF1042 domain and we show that C2 does not interact with another one of the DUF1042 domain-containing proteins. Our results suggest a role for SPATA4 in membrane traffic and a specialized function for TRAPP in spermatocytes.     

A genetic screen identifies novel non-compatible loxP sites

The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment. However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied. To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site. One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture. By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.     

Baculovirus-based genetic screen for antiapoptotic genes identifies a novel IAP

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. We have used this pro-apoptotic property of the p35 null virus to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. We report here the identification of Tn-IAP1, a novel member of the IAP family of cell death inhibitors. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources.     

Isolation of a mammalian homologue of a fission yeast differentiation regulator

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.     

A cytological procedure to screen mammalian temperature-sensitive mutants for cell-cycle-related defects.

Interphase cells stained with quinacrine dihydrochloride show distinctive fluorescent nuclear patterns according to their position in the cell cycle. These patterns were used to determine whether temperature-sensitive growth mutants of the Syrian hamster cell line BHK-21 are blocked at specific stages of the cell cycle. These cytological studies confirmed the previous conclusion that ts Af8 cell line derived from BHK cells is a GI cell-cycle mutant. The method promises to be of use as an initial probe in screening for cell-cycle mutants.     

A mammalian translation initiation factor can substitute for its yeast homologue in vivo

The translation initiation factor 4E (eIF-4E) is involved in the recognition of the cap structure at the 5' end of eukaryotic mRNA and facilitates ribosome binding. Subsequently, additional initiation factors mediate ribosomal scanning of mRNA and initiator AUG recognition (Shatkin, A. J. (1985) Cell 40, 223-224; Rhoads, R. E. (1988) Trends Biochem. Sci. 13, 52-56; Edery, I., Pelletier, J., and Sonenberg, N. (1987) in Translational Regulation of Gene Expression (Ilan, J., ed) pp. 335-366, Plenum Publishing Corp., New York). We show here that initiation factor 4E is functionally conserved between the unicellular eukaryote Saccharomyces cerevisiae and mammals. Although the amino acid identity of the factors from both species is limited to only 33%, mouse eIF-4E can substitute for yeast eIF-4E in vivo without major effects on cell viability, growth, and mating. This finding provides a starting point for new experimental strategies to investigate the structure-function relationship of eukaryotic translation initiation factor eIF-4E.     

A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing

The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1 mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of beta-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human, Caenorhabditis elegans, Arabidospis, and Drosophila.     

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.     

A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.