Abstract
Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.
Highlights
The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells, comprised of a complex network of sheets, tubules, junctions and contact sites that can occupy more than 35% of the entire cell volume (Valm et al, 2017) and a significant fraction of the total membrane surface area
With a design to determine cognate ER-resident E3s (ER-E3s) co-factors and substrates, we developed a generalised expression protocol coupled to a comparative immunoprecipitation liquid chromatography tandem mass spectrometry (IP-LC-MS/MS) workflow that enriched for proteins interacting at the ER
Comparative proteomic strategies have generated extensive and informative interaction networks of ubiquitination machinery (Sowa et al, 2009; Bennett et al, 2010). We have applied those principals to expand the functional ER-associated degradation (ERAD) modules described for Hrd1 and gp78/AMFR (Christianson et al, 2012) by mapping over 450 interactions to form the landscapes for 21 ER-E3s
Summary
The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells, comprised of a complex network of sheets, tubules, junctions and contact sites that can occupy more than 35% of the entire cell volume (Valm et al, 2017) and a significant fraction of the total membrane surface area. The continuous lattice it forms with the nuclear envelope (NE) makes extensive and dynamic contacts through distal projections with mitochondria (Kornmann et al, 2009), peroxisomes (Smith and Aitchison, 2013), endosomes (van der Kant and Neefjes, 2014; Honscher and Ungermann, 2014), plasma membrane (Saheki and De Camilli, 2017) and lipid droplets (reviewed in Wu et al, 2018a).
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