The human ribosome-associated complex suppresses prion formation in yeast.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

Systematic screen of Schizosaccharomyces pombe deletion collection uncovers parallel evolution of the phosphate signal transduction pathway in yeasts.

The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in Saccharomyces cerevisiae. We asked whether the PHO pathway was the same in the distantly related fission yeast Schizosaccharomyces pombe. We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of pho1(+) transcription. We identified a novel zinc finger-containing protein (encoded by spbc27b12.11c(+)), which we have named pho7(+), that is essential for pho1(+) transcriptional induction during phosphate starvation. Few of the S. cerevisiae genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in S. pombe. Only the most upstream genes in the PHO pathway in S. cerevisiae (ADO1, DDP1, and PPN1) share a similar role in both yeasts. Because ADO1 and DDP1 regulate ATP and IP(7) levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.

A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.

Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.

Prion-dependent proteome remodeling in response to environmental stress is modulated by prion variant and genetic background.

A number of fungal proteins are capable of adopting multiple alternative, self-perpetuating prion conformations. These prion variants are associated with functional alterations of the prion-forming protein and thus the generation of new, heritable traits that can be detrimental or beneficial. Here we sought to determine the extent to which the previously-reported ZnCl2-sensitivity trait of yeast harboring the [PSI+] prion is modulated by genetic background and prion variant, and whether this trait is accompanied by prion-dependent proteomic changes that could illuminate its physiological basis. We also examined the degree to which prion variant and genetic background influence other prion-dependent phenotypes. We found that ZnCl2 exposure not only reduces colony growth but also limits chronological lifespan of [PSI+] relative to [psi-] cells. This reduction in viability was observed for multiple prion variants in both the S288C and W303 genetic backgrounds. Quantitative proteomic analysis revealed that under exposure to ZnCl2 the expression of stress response proteins was elevated and the expression of proteins involved in energy metabolism was reduced in [PSI+] relative to [psi-] cells. These results suggest that cellular stress and slowed growth underlie the phenotypes we observed. More broadly, we found that prion variant and genetic background modulate prion-dependent changes in protein abundance and can profoundly impact viability in diverse environments. Thus, access to a constellation of prion variants combined with the accumulation of genetic variation together have the potential to substantially increase phenotypic diversity within a yeast population, and therefore to enhance its adaptation potential in changing environmental conditions.

The [PSI +] yeast prion does not wildly affect proteome composition whereas selective pressure exerted on [PSI +] cells can promote aneuploidy.

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI +] prion state leads to more translational read-through. Although extensive studies have been done on [PSI +], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi -] and [PSI +] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI +] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI +] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI +]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI +] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI +] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.

Thiostrepton-resistant mutants of Thermus thermophilus.

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

The ribosome-associated complex antagonizes prion formation in yeast.

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI(+)] prion - an alternative conformer of Sup35 protein - and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.

Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly.

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.