Lipid-gated vesicular trafficking directs HSPA1A to the plasma membrane through the endo-lysosomal network.

HSPA1A is a stress-inducible molecular chaperone that localizes to the plasma membrane (PM) of heat-shocked and cancer cells, where its membrane-associated form contributes to therapeutic resistance, membrane stabilization, and immune modulation. Because HSPA1A lacks a signal peptide, it does not follow the classical secretory pathway; instead, it reaches the PM through unconventional routes whose vesicular intermediates and intracellular lipid requirements remain largely undefined. Here, we show that following heat shock, HSPA1A undergoes coordinated redistribution across the endo-lysosomal network. It transiently associates with PI(3)P-enriched early endosomes, progresses through Rab4A- and Rab4B-positive recycling endosomes, and accumulates in LAMP1-positive lysosomes, while avoiding degradative and slow-recycling routes. Pharmacological inhibition of the ER-Golgi pathway did not affect HSPA1A's PM localization, while disruption of endosomal maturation and lysosomal function resulted in significant reductions. Heat shock drives a progressive increase in lysosomal Bis(monoacylglycero)phosphate (BMP) immunoreactivity, and pharmacological BMP accumulation increased PM-HSPA1A, whereas intracellular antibody-mediated BMP blockade reduced it, identifying BMP-enriched lysosomes as regulatory hubs that govern HSPA1A PM competence. Using a rapamycin-inducible compartment-specific phosphatase system, we further demonstrate that PI(4)P is required not only at the PM for final docking but within early endosomes, late endosomes, and lysosomes, establishing a distributed PI(4)P requirement across the endosomal network. Together, these findings define a lipid-gated vesicular trafficking mechanism for HSPA1A PM localization and identify lysosomal BMP and endosomal PI(4)P as additional regulatory layers relevant to cancer cells in which constitutive lipid remodeling may sustain membrane-associated HSPA1A and its pro-survival functions.