Mutant casein kinase I (Hrr25p/Kti14p) abrogates the G1 cell cycle arrest induced by <Emphasis Type="Italic"> Kluyveromyces lactis </Emphasis>zymocin in budding yeast

Zymocin, a toxic protein complex produced by Kluyveromyces lactis, inhibits cell cycle progression in Saccharomyces cerevisiae. In studying its action, a resistant mutant ( kti14-1) was found to express the tot-phenotype typical of totDelta cells, toxin target (TOT) mutants that are impaired in RNA polymerase II Elongator function. Phenotypic analysis of a kti14-1 tot3Delta double mutant revealed a functional link between KTI14 and TOT/Elongator. Unlike totDelta cells, the kti14-1 mutant is sensitive to the drug methylmethane sulfonate (MMS), indicating that, besides being affected in TOT function, kti14-1 cells are also compromised in DNA repair. Single-copy complementation identified HRR25, which codes for casein kinase I (CKI), as KTI14. Kinase-minus hrr25 mutations (K38A and T176I) conferred zymocin resistance, while deletion of the other yeast CKI genes ( YCK1-3) had no effect. A mutation in KTI14 that truncates the P/Q-rich C-terminus of Hrr25p also dissociates MMS sensitivity from zymocin resistance; this mutant is resistant to the toxin, but shows normal sensitivity to MMS. Thus, although kinase-minus mutations are sufficient to protect yeast cells from zymocin, toxicity is also dependent on the integrity of the C-terminal region of Hrr25p, which has been implicated in determining the substrate specificity or localization of Hrr25p.     

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.     

Elongator function depends on antagonistic regulation by casein kinase Hrr25 and protein phosphatase Sit4

In yeast, the role for the Elongator complex in tRNA anticodon modification is affected by phosphorylation of Elongator subunit Elp1. Thus, hyperphosphorylation of Elp1 due to inactivation of protein phosphatase Sit4 correlates with Elongator-minus phenotypes including resistance towards zymocin, a tRNase cleaving anticodons of Elongator-dependent tRNAs. Here we show that zymocin resistance of casein kinase hrr25 mutants associates with hypophosphorylation of Elp1 and that nonsense suppression by the Elongator-dependent SUP4 tRNA is abolished in hrr25 or sit4 mutants. Thus changes that perturb the evenly balanced ratio between hyper- and hypophosphorylated Elp1 forms present in wild-type cells lead to Elongator inactivation. Antagonistic roles for Hrr25 and Sit4 in Elongator function are further supported by our data that Sit4 inactivation is capable of restoring both zymocin sensitivity and normal ratios between the two Elp1 forms in hrr25 mutants. Hrr25 binds to Elongator in a fashion dependent on Elongator partner Kti12. Like sit4 mutants, overexpression of Kti12 triggers Elp1 hyperphosphorylation. Intriguingly, this effect of Kti12 is blocked by hrr25 mutations, which also show enhanced binding of Kti12 to Elongator. Collectively, our data suggest that rather than directly targeting Elp1, the Hrr25 kinase indirectly affects Elp1 phosphorylation states through control of Sit4-dependent dephosphorylation of Elp1.     

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.     

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.     

After chitin docking, toxicity of Kluyveromyces lactis zymocin requires Saccharomyces cerevisiae plasma membrane H+-ATPase

Zymocin, a three-subunit (alpha beta gamma) toxin complex from Kluyveromyces lactis, imposes a cell cycle block on Saccharomyces cerevisiae. Phenotypic analysis of the resistant kti10 mutant implies a membrane defect, suggesting that KTI10 represents a gene involved early in the zymocin response. Consistently, KTI10 is shown here to be allelic to PMA1 encoding H(+)-ATPase, a plasma membrane H(+) pump vital for membrane energization (Delta Psi). Like pma1 mutants, kti10 cells lose viability at low pH, indicating a pH homeostasis defect, and resist the antibiotic hygromycin B, uptake of which is known to be Pma1 and Delta Psi sensitive. Similar to kti10 cells, pma1 mutants with reported H(+) pump defects survive in the presence of exozymocin but do not resist endogenous expression of its lethal gamma-toxin subunit. Based on DNA sequence data, kti10 cells are predicted to produce a malfunctional Pma1 variant with expression levels that are normal. Intriguingly, zymocin protection of kti10 cells is suppressed by excess H(+), a scenario ineffective in bypassing resistance of chitin or toxin target mutants. Together with unaltered zymocin docking and gamma-toxin import events in kti10 cells, our data suggest that Pma1's role in zymocin action is likely to involve activation of gamma-toxin in a step following its cellular uptake.     

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.     

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.     

Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module

Kti11p is a small, highly conserved CSL zinc finger-containing protein found in many eukaryotes. It was first identified as one of the factors required for maintaining the sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Then, it was found to be identical to Dph3, a protein required for diphthamide biosynthesis on eEF-2, the target of diphtheria toxin and Pseudomonas exotoxin A, in both yeast and higher eukaryotes. Furthermore, Kti11p/Dph3 was found to physically interact with core-Elongator, ribosomal proteins, eEF-2, two other proteins required for diphthamide modification on eEF-2, and DelGEF. Here, we determined the solution structure of Kti11p using NMR, providing the first structure of the CSL-class zinc-binding protein family. We present the first experimental evidence that Kti11p can bind a single Zn(2+) ion by its four conserved cysteine residues. The major structure of Kti11p comprises a beta sandwich as well as an alpha helix. Moreover, a structure-based similarity search suggests that it represents a novel structure and may define a new family of the zinc ribbon fold group. Therefore, our work provides a molecular basis for further understanding the multiple functions of Kti11p/Dph3 in different biological processes.     

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.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

tRNA and protein methylase complexes mediate zymocin toxicity in yeast

Modification of Saccharomyces cerevisiae tRNA anticodons at the wobble uridine (U34) position is required for tRNA cleavage by the zymocin tRNase killer toxin from Kluyveromyces lactis . Hence, U34 modification defects including lack of the U34 tRNA methyltransferase Trm9 protect against tRNA cleavage and zymocin. Using zymocin as a tool, we have identified toxin-resistant mutations in TRM9 that are likely to affect the U34 methylation reaction. Most strikingly, C-terminal truncations in Trm9 abolish interaction with Trm112, a protein shown to individually purify with Lys9 and two more methylases, Trm11 and Mtq2. Downregulation of a GAL1-TRM112 allele protects against zymocin whereas LYS9 , TRM11 and MTQ2 are dosage suppressors of zymocin. Based on immune precipitation studies, the latter scenario correlates with competition for Trm112 and in excess, some of these Trm112 partners interfere with formation of the toxin-relevant Trm9·Trm112 complex. In contrast to trm11 Δ or lys9 Δ cells, trm112 Δ and mtq2 Δ null mutants are zymocin resistant. In line with the identified role that methylation of Sup45 by Mtq2 has for translation termination by the release factor dimer Sup45·Sup35, we observe that SUP45 overexpression and sup45 mutants suppress zymocin. Intriguingly, this suppression correlates with upregulated levels of tRNA species targeted by zymocin's tRNase activity.     

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator‐dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.     

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.