HspA1A is a cytosolic molecular chaperone essential for cellular homeostasis. HspA1A also localizes at the plasma membrane (PM) of tumor and stressed cells. However, it is currently unknown how this cytosolic protein translocates to the PM. Taking into account that HspA1A interacts with lipids, including phosphatidylserine (PS), and that lipids recruit proteins to the PM, we hypothesized that the interaction of HspA1A with PS allows the chaperone to localize at the PM. To test this hypothesis, we subjected cells 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 increased during recovery from non-apoptotic heat-shock. Next, we selectively reduced PS targets by overexpressing the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, and determined that HspA1A’s membrane localization was greatly reduced. 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. Implementation of a fluorescent PS analog, TopFluor-PS, established that PS co-localizes with HspA1A. Collectively, these results reveal that HspA1A’s PM localization and anchorage depend on its selective interaction with intracellular PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response.

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.

Molecular chaperones, particularly the 70-kDa heat shock proteins (Hsp70s), are key orchestrators of the cellular stress response. To perform their critical functions, Hsp70s require the presence of specific co-chaperones, which include nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. BAG-1 is one of these proteins that function in a wide range of cellular processes, including apoptosis, protein refolding, and degradation, as well as tumorigenesis. However, the origin of BAG-1 proteins and their evolution between and within species are mostly uncharacterized. This report investigated the macro- and micro-evolution of BAG-1 using orthologous sequences and single nucleotide polymorphisms (SNPs) to elucidate the evolution and understand how natural variation affects the cellular stress response. We first collected and analyzed several BAG-1 sequences across animals, plants, and fungi; mapped intron positions and phases; reconstructed phylogeny; and analyzed protein characteristics. These data indicated that BAG-1 originated before the animals, plants, and fungi split, yet most extant fungal species have lost BAG-1. Furthermore, although BAG-1’s structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional specialization within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP density is unequally distributed within the BAG1 gene, and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the BAG domain region, which is an indication of positive selection. To further explore this notion, we performed several biochemical assays and found that only one out of five mutations tested altered the major co-chaperone properties of BAG-1. These data collectively suggest that although the co-chaperone functions of BAG-1 are highly conserved and can probably tolerate several radical mutations, BAG-1 might have acquired specialized and potentially unexplored functions during the evolutionary process.