The Plasma Membrane Translocation of HspA1A, a Stress Inducible 70‐kDa Heat Shock Protein, Depends on its Interaction with Membrane Lipids

HspA1A is a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70). This protein is a molecular chaperone that plays important roles in cell survival. Normally, HSPA1A functions within the cytoplasm. Yet, in stressed and cancer cells HspA1A also localizes at the plasma membrane (PM). In cancer cells, in particular, the presence of HspA1A at the cell surface allows the tumor to increase invasiveness and develop resistance to radiation therapy. Therefore, blocking this protein from being at the surface of tumors is a promising therapeutic. However, the translocation pathway of HspA1A to the PM remains puzzling, because this protein lacks membrane localization signals. Based on the fact that HspA1A interacts with lipids, including phosphatidylserine (PS) and several mono‐phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HspA1A with these lipids allows the chaperone to localize at the PM. To test this hypothesis, we subjected different cell lines to mild heat‐shock and the PM‐localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that HspA1A’s membrane localization and embedding increased during recovery from non‐apoptotic heat‐shock. Next, we selectively reduced PS, PI(4)P, and PI(3)P targets by co‐transfecting HspA1A with known PS‐, PI(4)P‐, and PI(3)P‐ biosensors, and determined that HspA1A’s membrane localization was greatly reduced in all three cases. In contrast, the reduction of PI(4,5)P2 availability by overexpression of the PLCδ‐PH biosensor had minimal effects on HspA1A’s PM‐localization. Additionally, we manipulated the cellular lipid content using ionomycin, a calcium ionophore that activates PLC, and phenylarsine oxide (PAO), a PI4‐Kinase inhibitor, using several lipid‐biosensors as positive and negative controls. These experiments revealed that the PM localization of HspA1A was unaffected by ionomycin, but was greatly reduced in the presence of PAO, corroborating the findings obtained by the lipid‐biosensors on the importance of specific lipids in the PM translocation of HspA1A. Collectively, these findings strongly support the notion that HspA1A’s PM localization and anchorage is a complex lipid‐driven phenomenon controlled by the binding of the chaperone to endosomal and plasma membrane PS, PI(4)P, and PI(3)P. Our findings provide the basis for interventions that block HSPA1A’s translocation to the tumor cell’s PM allowing the cell to remain sensitive to radiation therapy and decrease its invasiveness. Support or Funding Information This project was supported by funds from NIH