Insertion of Hsp70 into Membranes is Mediated by Negative-Charged Phospholipids and Modulated by the Fluidity of the Bilayer

Abstract

The universal response to stress is mediated by the expression of heat shock proteins (hsp), which are involved in repair and recovery from the insult. In addition, their presence provides protection from subsequent insults, which has been coined stress tolerance. The cytoprotective role of hsp has been associated with their chaperone function within the cytosol. However, hsp were recently found outside cells where they acted as signaling molecules directed at activating the immune system to avoid the propagation of the insult. Hsp70, the major inducible form of the hsp family, does not contain any consensus secretory signal that predicts its secretion via the ER-Golgi pathway. Therefore, it is likely that the export of Hsp70 to the extracellular environment is mediated by the propose that Hsp70 is exported by a novel mechanism initiated by the translocation of the protein into the plasma membrane and is released associated with vesicles called export or extracellular vesicles (ECV). To test this hypothesis, we developed a liposome insertion assay using pure recombinant Hsp70. We found that Hsp70 insertion into lipid membranes was spontaneous and specific for negatively charged lipids, such as phosphatidylserine and phosphatidylglycerol. In contrast, positive or neutral lipids did not support membrane insertion. We also found that less fluid lipid environments highly favored membrane insertion, which resembles in vivo observations indicating the presence of Hsp70 in lipid rafts of cells. In summary, our observations support the hypothesis that membrane insertion is the first stage of secretion of Hsp70 into the extracellular environment in the form of vesicles. We speculate that hsp70-ECV may result in a robust activation of the immune system that is part of the systemic response to stress.Supported by NIH R01 GM098455.