Interaction between a 70‐kDA heat shock protein and phosphatidylserine in stressed human cells

Abstract

Seventy‐kilodalton heat shock proteins (Hsp70s) are molecular chaperones playing essential roles in protein homeostasis and cellular survival. Apart from their indispensable roles in cellular homeostasis specific Hsp70s also localize at the plasma membrane and bind to several lipids, including phosphatidylserine (PS). The interaction of Hsp70s with PS regulates important processes, like microautophagy and cell death. However, the conditions that trigger and promote the interaction between Hsp70s and intracellularly localized PS in human cells remain unknown. To shed light to this newly described property of Hsp70s, we tested whether heat‐shock, a stress known to affect both protein expression and membrane lipid composition, triggers the binding of HspA1A, a stress inducible Hsp70, to PS. To this end, we first determined whether HspA1A and the C2 domain of lactadherin (Lact‐C2), a known PS‐biosensor, compete for binding to intracellular PS. Specifically, HeLa cells were subjected to mild heat‐shock and the competition was assessed by quantifying the amount of membrane localized HspA1A in the presence or absence of the Lact‐C2 protein. These imaging experiments revealed that in the absence of Lact‐C2 HspA1A's membrane localization increases continuously after heat‐shock and reaches a maximal value at 8 hours during recovery. In the presence of Lact‐C2, however, HspA1A's membrane localization was minimal and did not show an increase during recovery after heat‐shock. These experiments were verified using cell surface biotinylation. In these experiments, Hek293 cells, after heat‐shock were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads, and the biotinylated proteins were analyzed by western blots and quantified using densitometry. Collectively, these results strongly suggest that HspA1A binds to intracellular PS, and that HspA1A's membrane localization and anchorage depends on its interaction with PS. These observations institute PS as a new and dynamic partner in the cellular stress response and establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.Support or Funding InformationThis project was supported by funds from NIH and CSUF to NN