WNK1 is an assembly factor for the human ER membrane protein complex

Abstract

The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

“…Kinase assay. Full-length human WNK1 (residues 1-2382;) was expressed with a C-terminal MYC-DDK (1xFLAG) tag from a pCMV6-Entry backbone (Origene, USA, RC218208) in Expi 293 F suspension cells (Thermo Fisher Scientific, A14527) 100 . Cells were grown at 37˚C, 8% CO 2 and shaking in 1 L roller bottles with vented caps.…”

Quality control of protein complex composition

“…Reminiscent of HUWE1, other E3 ligases bind excess subunits through surfaces occluded upon complex formation. MKRN recognizes a hydrophobic region in the EMC2 subunit of the ER membrane protein complex that is shielded by its partner EMC8 (Pleiner et al, 2021). In addition to ribosomal proteins, UBE2O detects unassembled a-globin by accessing a site that in hemoglobin is engaged by b-globin (Nguyen et al, 2017;Yanagitani et al, 2017).…”

“…Akin to the proteasome, many complexes require assembly factors for their biogenesis (Klinge and Woolford, 2019;Pleiner et al, 2021), and quality control E3 ligases frequently bind or regulate chaperones (Connell et al, 2001;Rodrigo-Brenni et al, 2014). HECTD1, which produces branched ubiquitin chains implicated in quality control (Harris et al, 2021;Yau et al, 2017), ejects the assembly factor ZNF622 from nascent ribosomes (Lv et al, 2021).…”

Large protein complex interfaces have evolved to promote cotranslational assembly

“…We focused on the analysis of cyclic homomers that do not localise to plasma or endomembrane systems, because of the competing hydrophobic forces exerted by protein-lipid interactions. Cotranslational membrane insertion of complexes is likely an active process requiring, for example, shielding factors 63,64 , rather than being solely driven by hydrophobic surface area. Despite the relatively limited number of structures available, we detect a significant difference in the mean subunit-to-subunit interface area among soluble members of the cyclic symmetry group (Figure 1B), with a 19% increase in the mean of cotranslationally forming members (p = 0.03, Wilcoxon rank-sum test).…”