Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.