Roland Klassen

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G1 phase of the cell cycle. Despite its heterotrimeric (αβγ) structure, intracellular expression of its smallest subunit, the γ‐toxin, is alone responsible for the G1 arrest. The α subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR‐mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3Δ), blocked export (chs7Δ) or reduced activation (chs4Δ), combined with mislocalization (chs4Δchs5Δ; chs4Δchs6Δ; chs4Δchs5Δchs6Δ) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII‐polymerized chitin, which amounts to almost 90% of the cells chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells. Copyright

The primary target of the killer toxin from Pichia acaciae is tRNAGln

The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S‐phase of the cell cycle and induces DNA double‐strand breaks (DSBs). Surprisingly, loss of the tRNA‐methyltransferase Trm9 – along with the Elongator complex involved in synthesis of 5‐methoxy‐carbonyl‐methyl (mcm5) modification in certain tRNAs – conferred resistance against PaT. Overexpression of mcm5‐modified tRNAs identified tRNAGln(UUG) as the intracellular target. Consistently, toxin‐challenged cells displayed reduced levels of tRNAGln and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNAGln(UUG). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNAGlu(UUC), affecting its target in a mcm5‐dependent manner, PaT exerts activity also on tRNAGln lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNAGln(UUG), tRNAGln(CUG) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNAGln(CUG) as an additional TRM9‐independent target, overexpression of PaTs tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.

Induction of DNA damage and apoptosis in Saccharomyces cerevisiae by a yeast killer toxin

The cellular response of Saccharomyces cerevisiae to a linear plasmid encoded killer toxin from Pichia acaciae was analysed. As for the Kluyveromyces lactis zymocin, such toxin was recently shown to bind to the target cells chitin and probably acts by facilitating the import of a toxin subunit. However, as distinct from zymocin, which arrests cells in G1, it provokes S‐phase arrest and concomitant DNA damage checkpoint activation. Here, we report that such novel toxin type causes cell death in a two‐step process. Within 4 h in toxin, viability of cells is immediately reduced to approximately 30%. Elevated mutation rates at the CAN1 locus prove DNA damaging mediated by the toxin. Cells arrested artificially in G1 or G2/M are very rapidly affected, while cells arrested in S loose their viability at a slower rate. S‐phase arrest is, thus, a response of target cells to cope with DNA damage induced by the toxin. A second decline in viability requiring metabolically active target cells emerges upon toxin exposure over 10 h. During this phase, toxin treated cells develop abnormal nuclear morphology and react positive to terminal deoxynucleotidyl transferase‐mediated nick end‐labelling (TUNEL), indicative of DNA fragmentation. Furthermore, as judged from staining with fluorescein conjugated annexinV, cells expose phosphatidylserine at the outer membrane face and the formation of reactive oxygen species (ROS) is increased. ROS formation and concomitant cell death was heavily suppressed in a rho‐ derivative of the tester strain, while immediate reduction of viability was indistinguishable from the wild type. As a strain lacking the cellular target because of defects in the major chitinsynthase (Chs3) did not display such characteristic changes, the chitin binding and DNA‐damaging P. acaciae toxin constitutes an apoptosis inducing protein. Both, DNA‐damaging and apoptosis induction are unique features of this novel toxin type.

Novel yeast killer toxins provoke S-phase arrest and DNA damage checkpoint activation ‡

Certain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host. Such killer plasmids (pPac1‐2 of P. acaciae and pWR1A of W. robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis. Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1. As for the latter, they encode functions mediating binding of the toxin to the target cells chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells. Consistently, mutations affecting the target cells major chitin synthase (Chs3) protect it from toxin action. Heterologous intracellular expression of respective open reading frames identified cell cycle‐arresting toxin subunits deviating structurally from the likewise imported γ‐subunit of the K. lactis zymocin. Accordingly, toxicity of both P. acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action. Thus, P. acaciae and Wingea toxins differ in their mode of action from the G1‐arresting zymocin. Fluorescence‐activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post‐G1 during the S phase. Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated. As a mutant carrying the checkpoint‐deficient allele rad53‐11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.

Linear Protein-Primed Replicating Plasmids in Eukaryotic Microbes

Linear plasmids of eukaryotic microbes are contemporary manifestations of ancient viruses, which have adjusted to two cellular compartments during evolution, i.e., to the mitochondrium or to the cytoplasm. In either case, infectious viral functions do not (any longer) exist. Mitochondrial as well as cytoplasmic elements display a minimized gene equipment and an archetypical mode of replication. Plasmids of filamentous fungi are selfish DNA elements routinely residing in the mitochondria, in which they underwent coevolution with their hosts. In addition to the archetypical viral B-type DNA polymerase, they typically exclusively encode a viral RNA polymerase. Usually, there are neither positive nor negative impacts on their hosts. In a few instances, however, symptoms redolent of a molecular disease, such as the accumulation of defective mitochondria and early onset of senescence, manifest in plasmid-harboring strains. Cytoplasmic localization, which applies for almost all yeast linear plasmids known so far, evidently enforced a more complex enzyme repertoire (of viral origin) to accomplish autonomous extranuclear and extramitochondrial replication and transcription, such as a helicase, ssDNA binding proteins, and a capping enzyme. Accompanying cytoplasmic plasmids relying functionally on an autonomous element are rather frequent; some encode protein toxins, which benefits the respective host while competing with other yeasts (killer phenotype). Such toxins assure autoselection of the plasmid system as well. Two distinct toxic principles are known up to the present: one was shown to be a tRNase, whereas the other clearly involves a DNA-damaging mode of action.

PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication

DNA polymerase δ (Polδ) plays an essential role in replication from yeast to humans. Polδ in Saccharomyces cerevisiae is comprised of three subunits, the catalytic subunit Pol3 and the accessory subunits Pol31 and Pol32. Yeast Polδ exhibits a very high processivity in synthesizing DNA with the proliferating cell nuclear antigen (PCNA) sliding clamp; however, it has remained unclear how Polδ binds PCNA to achieve its high processivity. Here we show that PCNA interacting protein (PIP) motifs in all three subunits contribute to PCNA-stimulated DNA synthesis by Polδ, and mutational inactivation of all three PIP motifs abrogates its ability to synthesize DNA with PCNA. Genetic analyses of mutations in these PIPs have revealed that in the absence of functional Pol32 PIP domain, PCNA binding by both the Pol3 and Pol31 subunits becomes essential for cell viability. Based on our biochemical and genetic studies we infer that yeast Polδ can simultaneously utilize all three PIP motifs during PCNA-dependent DNA synthesis, and suggest that Polδ binds the PCNA homotrimer via its three subunits. We consider the implications of these observations for Polδ’s role in DNA replication.

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNALys UUU, tRNAGlu UUC and tRNAGln UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALys UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALys UUU hypomodification and malfunction.

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype.

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.

The diphthamide modification pathway from Saccharomyces cerevisiae--revisited.

Diphthamide is a conserved modification in archaeal and eukaryal translation elongation factor 2 (EF2). Its name refers to the target function for diphtheria toxin, the disease‐causing agent that, through ADP ribosylation of diphthamide, causes irreversible inactivation of EF2 and cell death. Although this clearly emphasizes a pathobiological role for diphthamide, its physiological function is unclear, and precisely why cells need EF2 to contain diphthamide is hardly understood. Nonetheless, the conservation of diphthamide biosynthesis together with syndromes (i.e. ribosomal frame‐shifting, embryonic lethality, neurodegeneration and cancer) typical of mutant cells that cannot make it strongly suggests that diphthamide‐modified EF2 occupies an important and translation‐related role in cell proliferation and development. Whether this is structural and/or regulatory remains to be seen. However, recent progress in dissecting the diphthamide gene network (DPH1–DPH7) from the budding yeast Saccharomyces cerevisiae has significantly advanced our understanding of the mechanisms required to initiate and complete diphthamide synthesis on EF2. Here, we review recent developments in the field that not only have provided novel, previously overlooked and unexpected insights into the pathway and the biochemical players required for diphthamide synthesis but also are likely to foster innovative studies into the potential regulation of diphthamide, and importantly, its ill‐defined biological role.

Anticodon nuclease encoding virus-like elements in yeast.

A variety of yeast species are known to host systems of cytoplasmic linear dsDNA molecules that establish replication and transcription independent of the nucleus via self-encoded enzymes that are phylogenetically related to those encoded by true infective viruses. Such yeast virus-like elements (VLE) fall into two categories: autonomous VLEs encode all the essential functions for their inheritance, and additional, dependent VLEs, which may encode a toxin–antitoxin system, generally referred to as killer toxin and immunity. In the two cases studied in depth, killer toxin action relies on chitin binding and hydrophobic domains, together allowing a separate toxic subunit to sneak into the target cell. Mechanistically, the latter sabotages codon–anticodon interaction by endonucleolytic cleavage of specific tRNAs 3′ of the wobble nucleotide. This primary action provokes a number of downstream effects, including DNA damage accumulation, which contribute to the cell-killing efficiency and highlight the importance of proper transcript decoding capacity for other cellular processes than translation itself. Since wobble uridine modifications are crucial for efficient anticodon nuclease (ACNase) action of yeast killer toxins, the latter are valuable tools for the characterization of a surprisingly complex network regulating the addition of wobble base modifications in tRNA.