The Large Hsp70 Grp170 Binds to Unfolded Protein Substrates in Vivo with a Regulation Distinct from Conventional Hsp70s

Linda M Hendershot,Julia Behnke

doi:10.1074/jbc.m113.507491

Linda M Hendershot, Julia Behnke

Open Access

https://doi.org/10.1074/jbc.m113.507491

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Abstract

The Hsp70 superfamily is a ubiquitous chaperone class that includes conventional and large Hsp70s. BiP is the only conventional Hsp70 in the endoplasmic reticulum (ER) whose functions include: assisting protein folding, targeting misfolded proteins for degradation, and regulating the transducers of the unfolded protein response. The ER also possesses a single large Hsp70, the glucose-regulated protein of 170 kDa (Grp170). Like BiP it is an essential protein, but its cellular functions are not well understood. Here we show that Grp170 can bind directly to a variety of incompletely folded protein substrates in the ER, and as expected for a bona fide chaperone, it does not interact with folded secretory proteins. Our data demonstrate that Grp170 and BiP associate with similar molecular forms of two substrate proteins, but while BiP is released from unfolded substrates in the presence of ATP, Grp170 remains bound. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal α-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. We find that in the absence of the α-helical domain the interaction of Grp170 with substrates is reduced. In striking contrast, deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s.

Highlights

Large Hsp70s are structurally similar to conventional Hsp70s but functionally less well understood
Identification and Characterization of Direct glucose-regulated protein of 170 kDa (Grp170):Substrate Interactions in Vivo—Our understanding of the ability of large Hsp70 family members to bind to substrates is based on a combination of in vitro studies with purified proteins [13, 15,16,17,18, 41, 42] and co-immunoprecipitation with the multichaperone complexes formed in vivo (19 –23, 43)
The release of BiP from isolated complexes by ATP allowed us to demonstrate that Grp170 directly binds to several partially folded or unfolded endoplasmic reticulum (ER) substrates in vivo, extending in vitro studies with purified recombinant mammalian [17] and yeast [15] Grp170, as well as other cytosolic large Hsp70s [13, 16, 18, 44], which showed that all of them prevented the aggregation of denatured luciferase

Summary

Background

Large Hsp70s are structurally similar to conventional Hsp70s but functionally less well understood. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal ␣-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. Deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s. In the case of BiP, binding to incompletely folded proteins is regulated by its bound nucleotide, ATP or ADP, and by various ER-localized DnaJ family members (ERdjs), which can deliver substrates to BiP and connect it to the ER functions of protein synthesis, folding or degradation [4, 5]. Together our data reveal that Grp170 directly interacts with unfolded protein substrates in cells, but the regulation of its substrate binding function is strikingly different than for conventional Hsp70s

EXPERIMENTAL PROCEDURES

RESULTS

80 IP: FLAG

DISCUSSION