Abstract

Molecular chaperones and in particular the 70‐kD heat shock proteins (Hsp70s) are key regulators of the cellular stress response and alterations in their function have direct physiological consequences. To determine how natural mutations alter the function of Hsp70s we used single nucleotide polymorphisms (SNPs) found on HSPA1A the major stress inducible Hsp70 gene in humans. Specifically, the wild‐type (WT) HSPA1A sequence was subcloned into both bacterial and mammalian expression vectors and the mutated gene variants were generated using site directed mutagenesis. Recombinant proteins corresponding to the WT and mutated variants were then generated and tested for their ability to hydrolyze ATP. These experiments revealed that these mutations significantly change the rate by which the protein hydrolyzes ATP. The mutant proteins' ability to properly localize intracellularly within mammalian cells was also tested using immunofluorescence and confocal microscopy. These experiments demonstrated that the mutants and the WT protein had similar subcellular localization. However, live‐dead and cell proliferation assays revealed that some of these mutations significantly change cellular survival after heat stress. Given that these natural variants are either population‐specific or clinical we believe that the observed functional differences alter the ability of cells and the individuals carrying them to cope with stress or disease.

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