Biomedical subjects

Daniel Jablonowski

Publications and source records attributed to Daniel Jablonowski.

10 recordsLinked to original sources

PubMed2006

tRNAGlu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin-induced cell death in yeast.

Zymocin-induced cell death in Saccharomyces cerevisiae requires the toxin-target (TOT) effector Elongator, a protein complex with functions in transcription, exocytosis and tRNA modification. In line with the latter, trm9Delta cells lacking a tRNA methylase specific for wobble uridine (U(34)) residues survive zymocin and in excess, the Trm9 substrate tRNA(Glu) copies zymocin protection of Elongator mutants. Phenotypes typical of a tot3/elp3Delta Elongator mutant are absent from trm9Delta cells but copied in a tot3Deltatrm9Delta double mutant suggesting that Elongator acts upstream of Trm9. Consistent with Elongator-dependent tRNA modification being more important to mRNA decoding than Trm9, SUP4 and SOE1TRNA suppressors are highly sensitive to loss of Elongator and tRNA U(34) hypomodification. As Trm9 overexpression counteracts the effect of high-copy tRNA(Glu), zymocin suppression by high-copy tRNA(Glu) may reflect tRNA hypomethylation of trm9Delta cells. Thus, Trm9 methylation may enable recognition of tRNA by zymocin, a notion supported by a dramatic reduction of tRNA(Glu) levels in zymocin-treated cells and by cytotoxic zymocin residues conserved between bacterial nucleases and a tRNA modifying GTPase. In sum, Trm9 is a bona fideTOT pathway component whose methylation may be hijacked by zymocin to target tRNA function and eventually, mRNA translation.

Base Sequence↗

PubMed2006

Mating-type locus control of killer toxins from Kluyveromyces lactis and Pichia acaciae.

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.

Genes, Mating Type, Fungal↗

PubMed2006

Mutations in ABO1/ELO2, a subunit of holo-Elongator, increase abscisic acid sensitivity and drought tolerance in Arabidopsis thaliana.

The phytohormone abscisic acid (ABA) plays an important role in modulating plant growth, development, and stress responses. In a genetic screen for mutants with altered drought stress responses, we identified an ABA-overly sensitive mutant, the abo1 mutant, which showed a drought-resistant phenotype. The abo1 mutation enhances ABA-induced stomatal closing and increases ABA sensitivity in inhibiting seedling growth. abo1 mutants are more resistant to oxidative stress than the wild type and show reduced levels of transcripts of several stress- or ABA-responsive genes. Interestingly, the mutation also differentially modulates the development and growth of adjacent guard cells. Map-based cloning identified ABO1 as a new allele of ELO2, which encodes a homolog of Saccharomyces cerevisiae Iki3/Elp1/Tot1 and human IkappaB kinase-associated protein. Iki3/Elp1/Tot1 is the largest subunit of Elongator, a multifunctional complex with roles in transcription elongation, secretion, and tRNA modification. Ecotopic expression of plant ABO1/ELO2 in a tot1/elp1Delta yeast Elongator mutant complements resistance to zymocin, a yeast killer toxin complex, indicating that ABO1/ELO2 substitutes for the toxin-relevant function of yeast Elongator subunit Tot1/Elp1. Our results uncover crucial roles for ABO1/ELO2 in modulating ABA and drought responses in Arabidopsis thaliana.

Abscisic Acid↗

PubMed2005

Mannosyl-diinositolphospho-ceramide, the major yeast plasma membrane sphingolipid, governs toxicity of Kluyveromyces lactis zymocin.

Kluyveromyces lactis zymocin, a trimeric (alphabetagamma) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory gamma-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP)(2)C] synthase, which produces M(IP)(2)C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP)(2)C and sphingolipid mutants that have reduced levels of M(IP)(2)C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic gamma-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP)(2)C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.

Alleles↗

PubMed2004

The yeast elongator histone acetylase requires Sit4-dependent dephosphorylation for toxin-target capacity.

Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase. Here, we demonstrate that Elongator is a phospho-complex. Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action. Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190. Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation. Excess Sit4.Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4. Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin. Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Delta-nuclei retain Tot4. Together with the findings that sit4Delta and totDelta cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics.

Acetyltransferases↗

PubMed2003

Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification.

The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin-related system (URM1-UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.

Active Transport, Cell Nucleus↗

PubMed2002

Subunit communications crucial for the functional integrity of the yeast RNA polymerase II elongator (gamma-toxin target (TOT)) complex.

In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression. Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p). Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function. Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.

Acetyltransferases↗

PubMed2002

Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin.

The G(1) arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae cells requires a functional RNA polymerase II (pol II) Elongator complex. In studying a link between zymocin and pol II, progressively truncating the carboxyl-terminal domain (CTD) of pol II was found to result in zymocin hypersensitivity as did mutations in four different CTD kinase genes. Consistent with the notion that Elongator preferentially associates with hyperphosphorylated (II0) rather than hypophosphorylated (IIA) pol II, the II0/IIA ratio was imbalanced toward II0 on zymocin treatment and suggests zymocin affects pol II function, presumably in an Elongator-dependent manner. As judged from chromatin immunoprecipitations, zymocin-arrested cells were affected with regards to pol II binding to the ADH1 promotor and pol II transcription of the ADH1 gene. Thus, zymocin may interfere with pol II recycling, a scenario assumed to lead to down-regulation of pol II transcription and eventually causing the observed G(1) arrest.

Down-Regulation↗

PubMed2002

Genome organization of the linear Pichia etchellsii plasmid pPE1A: evidence for expression of an extracellular chitin-binding protein homologous to the alpha-subunit of the Kluyveromyces lactis killer toxin.

Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) is a non-killer yeast harbouring two cryptic linear cytoplasmic DNA-elements, pPE1A (6.7 kb) and pPE1B (12.8 kb). Cloning and complete sequencing of pPE1A revealed a 6749-bp element with a remarkably high A+T content of 77.6%. The termini of pPE1A were found to consist of inversely orientated identical nucleotide repetitions of 178bp, to which proteins are linked at the 5'-ends. It is only the second small, non-autonomous cytoplasmic yeast linear plasmid for which the complete nucleotide sequence is known. Five open reading frames (ORFs) were identified preceded by upstream conserved sequence motifs (UCS) characteristic for cytoplasmic promoters and perfectly matching the UCS consensus (ATNTGA). As none of the putative genes encodes a DNA-polymerase, pPE1A is the first yeast linear plasmid known that does not possess its own element-specific replication machinery. No function could be attributed to ORF1, 3, 4, and 5; the predicted ORF2 gene product is similar to chitin-binding proteins and chitinases, highest homologies were found to the precursor of the alpha- and beta-subunits of the secreted Kluyveromyces lactis zymocin. Consistently, the Orf2p could be isolated from the culture fluid by chitin-Sepharose affinity chromatography and characterized by immuno-probing with an antibody specific for the K. lactis killer toxin alpha-subunit. Production of the protein was found to be plasmid-dependent. The sequence of pPE1A has been submitted to the EMBL data library, Accession No. AJ409097.

Amino Acid Sequence↗

PubMed2002

Protein interactions within Saccharomyces cerevisiae Elongator, a complex essential for Kluyveromyces lactis zymocicity.

mTn3-tagging identified Kluyveromyces lactis zymocin target genes from Saccharomyces cerevisiae as TOT1-3/ELP1-3 coding for the RNA polymerase II (pol II) Elongator histone acetyltransferase (HAT) complex. tot phenotypes resulting from mTn3 tagging were similar to totDelta null alleles, suggesting loss of Elongator's integrity. Consistently, the Tot1-3/Elp1-3 proteins expressed from the mTn3-tagged genes were all predicted to be C-terminally truncated, lacking approximately 80% of Tot1p, five WD40 Tot2p repeats and two HAT motifs of Tot3p. Besides its role as a HAT, Tot3p assists subunit communication within Elongator by mediating Tot2-Tot4, Tot2-Tot5, Tot2-Tot1 and Tot4-Tot5 protein-protein interactions. TOT1 and TOT2 are essential for Tot4-Tot2 and Tot4-Tot3 interactions respectively. The latter was lost with a C-terminal Tot2p truncation; the former was affected by progressively truncating TOT1. Despite being dispensable for Tot4-Tot2 interaction, the extreme C-terminus of Tot1p may play a role in TOT/Elongator function, as its truncation confers zymocin resistance. Tot4p/Kti12p, an Elongator-associated factor, also interacted with pol II and could be immunoprecipitated while being bound to the ADH1 promoter. Two-hybrid analysis showed that Tot4p also interacts with Cdc19p, suggesting that Tot4p plays an additional role in concert with Cdc19p, perhaps co-ordinating cell growth with carbon source metabolism.

Adaptor Proteins, Signal Transducing↗