Lars Fichtner

Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin

Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin‐resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)‐associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts‐phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G1 cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Intriguingly, strains lacking non‐Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or non‐Elongator transcription elongation factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.

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 Elongators TOT function requires NLS‐ and karyopherin‐dependent nuclear import.

Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.

The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild‐type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator‐dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy‐terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.

KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin

The Kluyveromyces lactis zymocin and its γ‐toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin‐resistant kti11 and kti13 mutations using a single‐copy yeast library. Thus, we identified yeast open reading frames (ORFs) YBL071w‐A and YAL020c/ATS1 as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the γ‐toxin target site mutants, tot1–7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13, are actively transcribed protein‐encoding genes as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and in vivo HA epitope tagging. Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran–GTP/GDP cycle. Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression. This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function.

Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action.

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1–7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1–6). Unlike TOT1–3 (ELP1–3) and TOT5–7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co‐immunoprecipitation, and cell fractionation studies demonstrate partial co‐migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P‐loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P‐loop deletion renders Tot4p biologically inactive.

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

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 totΔ null alleles, suggesting loss of Elongators integrity. Consistently, the Tot1–3/Elp1–3 proteins expressed from the mTn3‐tagged genes were all predicted to be C‐terminally truncated, lacking ≈ 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.

Chitin-Binding Capability of the Zymocin Complex from Kluyveromyces lactis

To study the chitin-binding properties of the zymocin secreted by a K. lactis killer strain.

Phenotypic Analysis of the Kluyveromyces lactis Killer Phenomenon

To study the yeast-yeast interaction between a K. lactis killer strain and sensitive S. cerevisiae isolates.