Self-Association of Purified Reconstituted ER Luminal Spacer Climp63.

Jinghua Zhao,Junjie Hu

doi:10.3389/fcell.2020.00500

Abstract

Membranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is predicted to be highly helical, and it may form bridges between parallel membranes, regulating the abundance and width of ER sheets. Here, we purified the LD and full-length (FL) Climp63 to analyze their homotypic interactions. The N-terminal tagged LD formed low-order oligomers in solution, but was extremely aggregation-prone when the GST tag was removed. Purified FL Climp63 formed detectable but moderate interactions with both the FL protein and the LD. When Climp63 was reconstituted into proteoliposomes with its LD facing out, the homotypic interactions were retained and could be competed by soluble LD, though vesicle clustering was not observed. These results demonstrate a direct self-association of Climp63, supporting its role as an ER luminal spacer.

Highlights

The endoplasmic reticulum (ER) is composed by two interconnected morphological domains: tubules and sheets (Baumann and Walz, 2001; Shibata et al, 2006)
These results suggest that the luminal domain (LD) likely forms oligomers and/or adopts an extended configuration that shortens its retention time in the column compared to other globular proteins
Because GST forms dimers (Supplementary Figure S3A), but not trimers (Sacchetta et al, 1993), these results suggest that cytoskeleton-linking membrane protein 63 (Climp63) LD undergoes moderate self-association in addition to that by the GST

Summary

Introduction

The endoplasmic reticulum (ER) is composed by two interconnected morphological domains: tubules and sheets (Baumann and Walz, 2001; Shibata et al, 2006). Though the morphogenesis of the tubular ER network has been studied extensively (Shibata et al, 2009; Hu et al, 2011; Lin et al, 2012), little is known about how sheets are formed. Investigation of professional secretory cells, such as pancreatic cells and plasma cells that contain massive ER sheets, sheds light on key regulators of sheet formation (Shibata et al, 2010). Three ER membrane proteins, cytoskeleton-linking membrane protein 63 (Climp63), p180, and kinectin, have been identified as sheet-enriched proteins that determine ER sheet formation. P180 and kinectin, both of which potentially contain extensive cytosolic coiled coil domains, are thought to flatten ER membranes using these coiled coils. The presence of polysomes on ER membranes likely promotes sheet formation (Shibata et al, 2010).

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Conclusion