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Abstract:
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In 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 . |