Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae

Bio-protocol
Laura E KnightonAndrew W Truman

Abstract

Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as Nematostella vectensis NvHsp70A, B and D) are cloned into an appropriate expression plasmid. Next, these plasmids are transformed into ssa1-4Δ yeast [expressing native Ssa1 from an uracil-based (URA3) plasmid] which are subsequently cured of the original yeast on 5-Fluroorotic Acid (5-FOA). The resulting cells can be screened for a variety of phenotypes which match to the activity of well-studied cellular pathways.

References

Jan 1, 1987·Methods in Enzymology·J D BoekeG R Fink
Jan 20, 2011·Molecular and Cellular Biology·Himjyot JaiswalSabine Rospert
Jul 29, 2015·Data in Brief·Andrew W TrumanStephen J Kron
Apr 27, 2018·Cell Stress & Chaperones·Shawn J WallerAndrew W Truman
Apr 26, 2019·Current Genetics·Sarah K LotzAndrew W Truman
Jun 25, 2019·Journal of Proteomics·Laura E KnightonAndrew W Truman
Jun 30, 2019·Nature Reviews. Molecular Cell Biology·Rina RosenzweigBernd Bukau

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Citations

Jun 11, 2020·The Journal of Biological Chemistry· NitikaMatthias C Truttmann

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