Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

Abstract

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex.

Cell entry of a host-targeting protein of oomycetes requires gp96

“…4c ). During the re-uptake of gp96 with or without a ligand, gp96 interacts with sulfated sites of lectins (OS-9) and heparin/HSPG 35 , 58 . Hence, after treating cells with the sulfation inhibitor NaClO, the binding of gp96 to the cell surface is abolished 35 in the same way as the uptake of SpHtp3 into fish cells (Supplementary Fig.…”

Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

“…However, it remains elusive whether the ERAD lectins directly recognize folding states of potential ERAD substrates or whether they simply act as lectins in cooperation with ER chaperones such as BiP and GRP94. Recently, two independent groups reported the in vitro interaction mode between OS-9 and GRP94 and the in vivo function of their complex [ 72 , 73 ]. In the former report, the disordered C-terminal segment of OS-9, including a mammalian-specific insertion following the MRH domain, was involved in the interaction over the middle and C-terminal domains of GRP94, suggesting cooperative interplay between these two proteins in the motionally flexible complex [ 72 ].…”

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

“…Data points with a high y:x ratio represent mRNAs specifically captured by the bait of interest. The former group codes for multiple EPHA family members, as well as HECTD1 and OS9, a lectin that localizes to the endoplasmic reticulum and binds partially folded proteins ( Seidler et al, 2014 ).…”