The relation of a-synuclein (aS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated aS species have in neurotoxicity in vivo, we generated aS variants by a structurebased rational design. Biophysical analysis revealed that the aS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar aS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between aS aggregates with impaired b-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric aS species and their in vivo function.

SummaryThe 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.

SummaryThe 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/Elongatordependent manner, global poly(A) 1 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.

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.

Summary 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.

KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin tional expression of its g-subunit (or g-toxin) is sufficient to mimic this arrest (Butler et al., 1991b). The a-and bsubunits are likely to be required for zymocin docking, a process involving interaction with cell wall chitin (Butler et al., 1991c;Jablonowski et al., 2001a).Zymocin-resistant S. cerevisiae mutants have been isolated (skt: Kawamoto et al., 1990; kti: Butler et al., 1994; iki: Kishida et al., 1996; tot: Frohloff et al., 2001). Ten distinct kti class II complementation groups suggest that a number of proteins participate in the process blocked by zymocin by either acting in a pathway or forming a multiprotein target (Butler et al., 1994). In favour of the latter, analysis of the tot mutants identified the six-subunit RNA polymerase II (RNAPII) Elongator as TOT, the putative g-toxin target complex (Frohloff et al., 2001;Jablonowski et al., 2001b;Winkler et al., 2001). Mutations in the TOT genes, six of which are identical to Elongator genes ELP1-6, lead to zymocin resistance (Yajima et al., 1997;Otero et al., 1999;Wittschieben et al., 1999;Fellows et al., 2000;Frohloff et al., 2001;Jablonowski et al., 2001b;Krogan and Greenblatt, 2001;Li et al., 2001;Winkler et al., 2001). The histone acetyltransferase (HAT) activity encoded by ELP3 (TOT3) is essential for both Elongator function in vivo and K. lactis g-toxin action (Wittschieben et al., 2000;Frohloff et al., 2001;Winkler et al., 2001). TOT3 mutagenesis revealed that zymocin sensitivity can be uncoupled from Elongator function, indicating that TOT interacts genetically with zymocin in a manner dependent on Elongator's HAT (Jablonowski et al., 2001b). Thus, TOT/Elongator appears to be the key player in K. lactis zymocicity. Cells lacking the Sit4p phosphatase (Sutton et al., 1991) display zymocin resistance and all other tot phenotypes, suggesting that TOT function may be a Sit4p-dependent process (Jablonowski et al., 2001c). As Elongator is dispensable for life and the g-toxin arrest is TOT dependent, the K. lactis zymocin cannot simply block TOT/Elongator function. Instead, zymocin may rather modify Elongator function, so that RNAPII activity becomes poisoned (Frohloff et al., 2001;Jablonowski et al., 2001b).To study the relationship between uncharacterized KTI genes and TOT/Elongator function, we identified KTI11 (YBL071w-A) and KTI13 (YAL020c/ATS1). KTI11 and KTI13 deletions confer a tot phenotype in common with tot1-7D cells including zymocin resistance, slow growth, Molecular Microbiology (2002) SummaryThe Kluyveromyces lactis zymocin and its g-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 KTI1...

Cell–cell and cell–surface adherence represents initial steps in forming multicellular aggregates or in establishing cell–surface interactions. The commonly used Saccharomyces cerevisiae laboratory strain S288c carries a flo8 mutation, and is only able to express the flocculin-encoding genes FLO1 and FLO11, when FLO8 is restored. We show here that the two flocculin genes exhibit differences in regulation to execute distinct functions under various environmental conditions. In contrast to the laboratory strain Σ1278b, haploids of the S288c genetic background require FLO1 for cell–cell and cell–substrate adhesion, whereas FLO11 is required for pseudohyphae formation of diploids. In contrast to FLO11, FLO1 repression requires the Sin4p mediator tail component, but is independent of the repressor Sfl1p. FLO1 regulation also differs from FLO11, because it requires neither the KSS1 MAP kinase cascade nor the pathways which lead to the transcription factors Gcn4p or Msn1p. The protein kinase A pathway and the transcription factors Flo8p and Mss11p are the major regulators for FLO1 expression. Therefore, S. cerevisiae is prepared to simultaneously express two genes of its otherwise silenced FLO reservoir resulting in an appropriate cellular surface for different environments.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G 1 phase of the cell cycle. Despite its heterotrimeric (abc) structure, intracellular expression of its smallest subunit, the c-toxin, is alone responsible for the G 1 arrest. The a 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 (chs3D), blocked export (chs7D) or reduced activation (chs4D), combined with mislocalization (chs4Dchs5D; chs4Dchs6D; chs4Dchs5Dchs6D) 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.