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 cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells. Copyright © 2001 John Wiley & Sons, Ltd.

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.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct⁶A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct⁶A37 or Ψ38/39 and mcm⁵U34 or s²U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNA^Lys_UUU or tRNA^Gln_UUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm⁵U34 or s²U34, and this defect can be rescued by overexpression of tRNA^Gln_UUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Summary: 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 tRNA^Gln_(UUG) as the intracellular target. Consistently, toxin‐challenged cells displayed reduced levels of tRNA^Gln and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA^Gln_(UUG). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA^Glu_(UUC), affecting its target in a mcm⁵‐dependent manner, PaT exerts activity also on tRNA^Gln 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 tRNA^Gln_(UUG), tRNA^Gln_(CUG) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA^Gln_(CUG) as an additional TRM9‐independent target, overexpression of PaT's 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.