Terminally misfolded or otherwise damaged proteins in the endoplasmic reticulum (ER) are degraded by the proteasome in a process termed endoplasmic reticulum associated degradation (ERAD). To reach the proteasome located in the cytosol, cargo proteins need to be dislocated across the ER lipid bilayer. Retrograde transport is realized by a set of membrane-spanning multi-protein complexes that are notoriously difficult to study: Despite decades of genetic and biochemical analysis of this dislocation event, there is little consensus regarding its molecular mechanism. Here, we characterize the dynamic assembly of the mammalian Hrd1-complex, one of the most thoroughly studied mammalian dislocons, using in situ multicolor single-particle tracking. Surprisingly, quantitative dual-color tracking reveals that the majority of Hrd1 is assembled into stable homo-tetramers in situ. A herein developed single-molecule assay based on binding competition we show that this tetramerization is driven by a short cytosolic stretch (Hrd1 479-530 , HAF-H) within the cytosolic domain of Hrd1. Combining classical purification assays, quantitative imagining, and structural predictions demonstrates that Hrd1 479-530 forms a highly stable tetrameric helix bundle via a conserved hydrophobic coiled-coil motif. While higher order assemblies of Hrd1 have been previously implicated, this work offers direct evidence of their role in dislocation, demonstrating that quantitative single-molecule imaging can yield information on species previously hidden for classical biochemistry.
Abel et al. (2026) studied this question.