Browsing by Subject "Saccharomyces"
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Item Evaluation of protein aggregation and organismal fitness(2011-05) Stovall, Gwendolyn Motz; Ellington, Andrew D.; Marcotte, Edward; Whiteley, Marvin; Wilke, Claus O.; Willets, KatherineIn quiescent yeast, the widespread reorganization of cytosolic proteins into punctate has been observed (Narayanaswamy et al. 2009). We seek to better understand and describe this reorganization, which we hypothesize to be a protein aggregation phenomenon. To test this hypothesis, we examined mutant proteins (Ade4p protein variants) in yeast with predicted non-native aggregation propensities and measured their punctate formation kinetics. Monitoring punctate formation kinetics involved the validation of an automated quantification technique using an Amnis ImageStream imaging flow cytometer. The automated punctate counts were strongly correlated with the manual punctate counts, with usual R² values of 0.99 or better, but evaluated 50-fold more cells per run. Fitness evaluations of the mutant yeast in the form of growth curves and batch competition experiments revealed the slowed growth of the Ade4-1286 strain and the functional inequality to the wild type strain of the Ade4-mtoin2034, Ade4-mtoin2105, and Ade4-2800 strains in competition experiments, especially when the mutants were forced to generate their own adenine. Subsequent structural analysis of the mutant proteins revealed destabilizing mutations for 4 of the 6 mutant proteins with 2 of the mutations classified as significantly destabilizing ([delta][delta]G >2 kcal/mol). We concluded that the reduction in protein fitness was likely due to the destabilizing effects of the mutations. Evaluation of the punctate formation kinetics revealed little difference between strains in the rate of punctate formation. Further examination revealed the wild type Ade4p and all of the mutants (with the exception of the Ade4-1286 mutant) were predicted to have similar aggregation propensities according to a secondary aggregation predicting algorithm (Zyggregator, Pawar et al. 2005). Additionally, solvent accessibility calculations estimate ~3-19% of the side chain surface area to be solvent accessible, which indicates proximity of mutations to the protein surface. However, mutating buried amino acids likely would have generated a greater disturbance (Matthews 1993, Tokuriki et al. 2007). We concluded that the mutations, although destabilizing, altered the aggregation propensity very little. Deletion of chaperone proteins (Hsp82p, Hsc82p, and Ssa1p) revealed no difference in the Ade4-GFP punctate formation kinetics, although a slight kinetic difference was detected in the chaperone (Hsp82p) knockout, Gln1-GFP strain and the wild type strain. While further workup is necessary in the chaperone knockout, Gln1-GFP work, the initial results are promising and suggest the involvement of protein folding machinery in punctate formation.Item Functional interactions of chromosome segregation factors with the 2 micron plasmid : possible evolutionary link between the plasmid portioning locus and the budding yeast centromere(2011-05) Huang, Chu-Chun; Jayaram, Makkuni; Dean, Appling R.; Arlen, Johnson W.; Paull, Tanya T.; Stevens, Scott W.The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence.