Exploring the Functional Complementation between Grp94 and Hsp90.

Kevin A Maharaj,Nanette L S Que,Shuang Wu,Daniel T Gewirth,Feng Hong,John D Huck,Zihai Li,Sabrina K Gill,Didier Picard

doi:10.1371/journal.pone.0166271

Abstract

Grp94 and Hsp90 are the ER and cytoplasmic paralog members, respectively, of the hsp90 family of molecular chaperones. The structural and biochemical differences between Hsp90 and Grp94 that allow each paralog to efficiently chaperone its particular set of clients are poorly understood. The two paralogs exhibit a high degree of sequence similarity, yet also display significant differences in their quaternary conformations and ATPase activity. In order to identify the structural elements that distinguish Grp94 from Hsp90, we characterized the similarities and differences between the two proteins by testing the ability of Hsp90/Grp94 chimeras to functionally substitute for the wild-type chaperones in vivo. We show that the N-terminal domain or the combination of the second lobe of the Middle domain plus the C-terminal domain of Grp94 can functionally substitute for their yeast Hsp90 counterparts but that the equivalent Hsp90 domains cannot functionally replace their counterparts in Grp94. These results also identify the interface between the Middle and C-terminal domains as an important structural unit within the Hsp90 family.

Highlights

The hsp90 family of molecular chaperones are essential for the survival of all eukaryotes
All constructs contained N-terminal hexahistidine tags and were constitutively expressed from a Trp marked p414GPD plasmid that has been previously shown to accumulate Hsp90 to near wildtype levels [27, 34]. These plasmids were introduced into yeast strain ECU82a, which lacks both chromosomal copies of Hsp90 and instead expresses wild-type yeast Hsp90 from a 2 μm URA3 marked plasmid [28]
Upon selection with 5-FOA, the URA3 plasmid is expelled, leaving the Hsp90 or chimeric construct expressed from the p414GPD plasmid as the sole Hsp90 in the cell. p414GPD plasmids expressing wild type yeast Hsp90 (1–709) and human Hsp90α (1–732) served as positive growth controls, while a plasmid encoding the yeast Hsp90 Ndomain (1–273) served as a negative growth control

Summary

Introduction

The hsp family of molecular chaperones are essential for the survival of all eukaryotes. They are among the most abundant proteins in the cell, comprising over 2% of the total polypeptide mass, and are key responders to a variety of stress signals which include heat shock, glucose deprivation, ATP depletion, and oxidative stress [1,2,3,4,5]. Perhaps reflecting its more specialized role, the set of clients for Grp is far smaller and consists of proteins destined for secretion or surface expression, including the Toll-like receptors (TLRs) [4, 9], integrins [4], IgG [10, 11], insulin-like growth factors (IGF-I, IGF-II) [12], Wnt co-receptor LRP6[13] and cell surface TGFβ–anchoring molecule GARP [14].

Methods

Results

Conclusion