Abstract 1096 Deciphering the Role of Lipids in Regulating Plasma Membrane and Extracellular HSPA1A

Heat shock protein A1A (HSPA1A) translocates to the plasma membrane (PM) and is also found extracellularly in both heat-shocked and cancer cells. This dual localization of HSPA1A, both on the PM and in the extracellular space, has inspired predictions of a connection between the two pools, suggesting that PM-bound HSPA1A (mHSPA1A) may be exported. However, these predictions remain unproven and the intriguing relationship between membrane bound and extracellular HSPA1A (eHSPA1A) remains undefined. Based on the fact that lipids regulate mHSPA1A localization, we hypothesized that lipid depletion would consequently prevent HSPA1A's export. To test this hypothesis, we combined pharmacological inhibition of key enzymes involved in lipid biosynthesis with lipid sequestration using biosensor proteins containing high-affinity lipid-binding domains. These complementary strategies enabled us to achieve potent lipid depletion and disruption. We quantified eHSPA1A through western blot analysis of conditioned media post heat shock and lipid depletion. Additionally, we harnessed the HiBit® luciferase-based system, allowing for simultaneous quantification of eHSPA1A, mHSPA1A, and cytosolic HSPA1A under various experimental conditions. Surprisingly, our findings consistently demonstrated that lipid depletion effectively blocked mHSPA1A localization while leaving eHSPA1A unaffected. This revelation contradicts current predictions, suggesting that mHSPA1A and eHSPA1A are independently regulated entities. Our findings reshape the prevailing model of HSPA1A trafficking, pointing towards the existence of separate mechanisms governing PM localization versus extracellular export. This paradigm shift has far-reaching implications for our understanding of HSPA1A's pivotal roles in cellular stress responses and cancer progression, where both membrane and extracellular HSPA1A play crucial yet paradoxical roles. Funds from NIH supported this project