Insertion of Hsp70 Into Membranes Correlates With the Flipping of Phosphatidlyserine Across the Lipid Bilayer

Abstract

Expression of heat shock proteins is the primary event in the cellular response to stress. Indeed, these proteins are crucial in preventing cell death and recovery after different physiological and environmental stresses. Hsp70 (Hsp72), which is the major stress-inducible member of the heat shock protein family, is primarily located in the cytosol. However, recent evidence has shown that this protein can be detected on the cell surface of transformed cells inserted into the plasma membrane. Hsp70 does not contain any hydrophobic domains that could predict its insertion into membranes. Consequently, the possible mechanism for translocation into membranes is likely a non-classical process. Pure recombinant Hsp70 was incubated with phosphatidylserine (PS) liposomes, and a concentration-dependent incorporation of the protein into the bilayer was observed. On the contrary, Hsp70 did not get incorporated into phosphatidylcholine (PC) liposomes. Liposomes made of a PS:PC mixture showed that insertion of Hsp70 into the bilayer was proportional to the PS concentration. In contrast, Hsp90 did not incorporate into PS liposomes. Hsp70 was found integrated into the lipid bilayer as demonstrated by lack of extraction by sodium carbonate or sonication treatment. Hsp70 inserted into PS liposomes could only be solubilized by non-ionic detergents. In cells, the presence of Hsp70 on the plasma membrane correlates with the flipping of PS to the outside of the membrane. These results demonstrate that Hsp70, which does not contain a predictable hydrophobic trans-membrane region, can spontaneously get inserted into a lipid environment by a process that may require the traslocation of PS across the lipid bilayer.