Membrane Localization of HspA1A, a Stress Inducible 70-kDa Heat-Shock Protein, Depends on Its Interaction with Intracellular Phosphatidylserine.

Bilog Bilog,Labanieh Labanieh,Nikolaidis Nikolaidis,Zapanta Zapanta,Oliverio Oliverio,Stahelin Stahelin,Smulders Smulders

doi:10.3390/biom9040152

Bilog Bilog, Labanieh Labanieh + Show 5 more

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https://doi.org/10.3390/biom9040152

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Abstract

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.

Highlights

HspA1A is the major stress inducible seventy-kilodalton heat-shock protein (Hsp70) in humans and mice, and is essential for cellular homeostasis and survival under both stress and pathological conditions [1]
In addition to its critical functions as a cytosolic molecular chaperone, HspA1A is found at the plasma membrane (PM) of stressed and cancer cells [2,3]
We provide below the measurement of the corrected total cell fluorescence (CTCF) values from images containing HspA1A-green fluorescence protein (GFP), WGA-AF555, and DAPI

Summary

Introduction

HspA1A is the major stress inducible seventy-kilodalton heat-shock protein (Hsp70) in humans and mice, and is essential for cellular homeostasis and survival under both stress and pathological conditions [1]. HspA1A does not contain known transmembrane or secretory signals and its presence at the PM and the extracellular medium has been puzzling Towards elucidating this conundrum, several reports revealed that HspA1A does not follow the classical PM-localization and secretion. The interaction of HspA1A with lipid rafts, which suggests a lipid-mediated anchoring mechanism, is further supported by ex vivo and in vitro studies revealing the interaction of HspA1A with specific membrane lipids [5,7,11,12,13,14,15,16,17,18,19] These lipids include among others globotriaosylceramide (Gb3), a lipid found primarily in lipid-rafts [7], and phosphatidylserine (PS), a lipid found primarily in the inner leaflet of the PM during normal cell growth [5,12,13,15,16,18,20]

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