Tutar, Yusuf Cumhuriyet University - School of Medicine - Department of Biochemistry, Turkey

Ssa1p function in the presence of nucleotide and Ssa1p oligomeric properties

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Aim. To investigate S. cerevisiae cytosolic Hsp70-Ssa1 protein thermal unfolding in the presence of ATP and a substrate protein A7. Method. Plasmid preparation was done according to the standard protocols. pC210 was used to overexpress Ssa1p and Ssa1-A17V. Plasmids were linearized and integrated in Pichiapastoris (strain GS 115) genome. Transformants were selected by YPD/zeocin/sorbitol plates. Harvested cells were lysed by silica beads and centrifuged to homogenity. Lysate was purified by following chromatographic purification. Nucleotide free Ssa1 protein was obtained by using Sephadex G-50 column. Gel filtration experiments on binding and aggregation studies were performed by using Sephacryl-200 resin and one meter column. Fluorescence experiments were performed with Shimadzu RF 450 spectrofluorometer. Results. Both Ssa1p and Ssa1-A17V showed no significant difference in substrate protein binding. The binding to peptide reaches a maximum around 40 ○C and then a sharp decrease was seen. In the absence of nucleotides, both proteins form monomeric species between 20-40ºC. Above oligomeric forms were observed. However, in the presence of 1 mM ATP oligomerization starts around 60ºC. Increase in temperature did not result in a second transition but a decrease in fluorescence was observed for both proteins. Conclusion. A7 and nucleotide ATP alters Ssa1p oligomeric properties and stability. This result is consistent with physiological conditions in a cell and explains how a cell survive under mild stress conditions through expressing Hsp70s.

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Hsp70 , S. erevisiae , Ssa1p

Cumhuriyet Medical Journal

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