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Biochemical characterization of the interaction between HspA1A and phospholipids.

ABSTRACT: Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

SUBMITTER: McCallister C

PROVIDER: S-EPMC4679732 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Biochemical characterization of the interaction between HspA1A and phospholipids.

McCallister Chelsea C Kdeiss Brianna B Nikolaidis Nikolas N

Cell stress & chaperones 20150905 1

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacteriz ...[more]

PMID: 26342809

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Project description:We have previously identified the scaffold protein liprin-?1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-?1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-?1 partner GIT1 in liprin-?1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-?1-enhanced spreading. Conversely inhibition of the formation of the liprin-?1-GIT complex by expression of liprin-?CC3 could still enhance spreading, although to a lesser extent compared to full length liprin-?1. No cumulative effects were observed after depletion of both liprin-?1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-?1 may compete with paxillin for binding to GIT1, while binding of ?PIX to GIT1 was unaffected by the presence of liprin-?1. Interestingly, GIT and liprin-?1 reciprocally regulated their subcellular localization, since liprin-?1 overexpression, but not the GIT binding-defective liprin-?CC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-?1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-?1-enhanced haptotatic migration, although the direct interaction between liprin-?1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-?1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-?1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-?1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

Dataset Information

Biochemical characterization of the interaction between HspA1A and phospholipids.

Publications

Biochemical characterization of the interaction between HspA1A and phospholipids.

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