Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins

Abstract

During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome1,2,3,4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination5,6,7,8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1’s RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.

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Fig. 1: Electrophysiological characterization of Hrd1 gating dynamics following auto-ubiquitination.
Fig. 2: Interaction of substrate with ubiquitinated Hrd1 stimulates channel activity.
Fig. 3: Binding of CPY* to the luminal and cytosolic sides of Hrd1.
Fig. 4: Auto-ubiquitination in the RING domain of Hrd1 is essential for channel stability, efficient substrate binding and ubiquitination.

Data availability

Source data for Figs. 1–4 and Extended Data Figs. 1–3 are provided with the paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank O. D. Bello and J. E. Rothman for providing the construct for ApoE422K, I. Bickmeyer and N. Nupur for technical assistance and T. Rapoport and R. Jahn for comments on the manuscript. This work was supported by the European Research Council (ERC) under the Horizon2020 research and innovation programme (grant no. 677770) to A.S., by the Deutsche Forschungsgemeinschaft SFB1190, grant nos. P12 (to M.M.) and P15 (to A.S.), and a Boehringer Ingelheim Fonds PhD Fellowship (to V.V.).

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A.S. and M.M. conceived the experiments and wrote the manuscript. V.V., N.D., D.R. and A.S. carried out the experiments: N.D. performed the electrophysiology; V.V. and A.S. performed the biochemistry; C.C.S. provided reagents and established the Ubc6-related experiments; and D.R. performed the electron microscopy. All authors contributed to data analysis.

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Correspondence to Alexander Stein or Michael Meinecke.

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Extended data

Extended Data Fig. 1 Characterization of Hrd1 liposomes and channel properties.

a, Liposomes containing C-terminally fluorescently labelled Hrd1 were floated in a Nycodenz step gradient. The gradient was fractionated and samples were analyzed by SDS-PAGE and fluorescence scanning. b, Fluorescently labeled Hrd1 in liposomes were incubated with Tobacco Etch Virus (TEV) protease that cleaves off the C-terminal SBP tag and the fluorescent dye. As a control, detergent-solubilized liposomes were incubated with TEV protease. Samples were analyzed by SDS PAGE and fluorescence scanning. c, Conductance state histogram zoom plot of Fig. 1e. The arrow and number indicate the highest observed conductance state for ubiquitinated Hrd1. To focus on large conductance states, the zoom plot starts at 100 pS. d, Current-voltage relationship of ubiquitinated Hrd1 at asymmetric salt. Arrows indicate the various reversal potentials and the numbers give the corresponding relative selectivities of potassium over chloride as calculated from the Goldman-Hodgkin-Katz equation. The red line represents linear regression (least-squares) of indicated data regions (length of red line on x-axis). Shown is a representative trace of three independent experiments. e, Time course of deubiquitination of Hrd1 using indicated concentrations of Usp2. Liposomes containing fluorescently labeled Hrd1 were immobilized onto streptavidin magnetic beads and incubated with ubiquitination mix in the presence or absence of ATP. After washing, Hrd1 liposomes were eluted with 2 mM biotin and incubated with the indicated amount of Usp2. The reaction was stopped by addition of SDS sample buffer. Samples were analyzed by SDS-PAGE and fluorescence scanning. This particular experiment was performed once. Other related deubiquitination experiments are shown in Fig. 3a and Extended Data Fig. 3a. In a-b, representative images of three independent experiments are shown. Source data and unprocessed gels are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 Interaction of misfolded substrates with Hrd1 reconstituted in liposomes or nanodiscs.

a, PrA*, but not PrA WT interacts with ubiquitinated Hrd1. Indicated amounts of Hrd1 liposomes were immobilized onto streptavidin magnetic beads via the C-terminal SBP tag on Hrd1. After incubation with ubiquitination mix in the presence or absence of ATP, beads were washed and incubated with 20 nM PrA* or 20 nM PrA WT at the indicated Hrd1 concentrations. The fraction of bound PrA* or PrA WT was determined from the supernatants. mean ± s.d (n = 3 independent experiments). b, Negative stain electron micrographs of glutaraldehyde-fixed Hrd1 nanodiscs. c, Size distribution from n = 738 Hrd1 nanodiscs. d, CPY*, but not CPY WT is efficiently ubiquitinated when added to the outside of Hrd1 in liposomes. Left: Fluorescently labelled CPY* or CPY WT (100 nM) was added to liposomes containing Hrd1 (200 nM) and incubated with ubiquitination mix with or without ATP. Samples from indicated time points were analyzed by SDS-PAGE and fluorescence scanning. Right: Quantification of three ubiquitination experiments. These data are also presented in Fig. 3h (CPY* lipos) and Extended Data Fig. 3c, d (CPY WT, WT Hrd1). mean ± s.d (n = 3 independent experiments). e, PrA*, but not PrA WT is efficiently ubiquitinated when added to the outside of Hrd1 in liposomes. Left: Fluorescently labelled PrA* or PrA WT (100 nM) was added to liposomes containing Hrd1 (200 nM) and incubated with ubiquitination mix with or without ATP. Samples from indicated time points were analyzed by SDS-PAGE and fluorescence scanning. Right: Quantification of three ubiquitination experiments. mean ± s.d (n = 3 independent experiments). Source data and unprocessed gels are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Characterization of the interaction of CPY* with autoubiquitinated Hrd1.

a, Release of CPY* from liposomes containing ubiquitinated Ubc6 or Hrd1 upon deubiquitination. Beads with immobilized liposomes containing ubiquitinated Hrd1 (250 nM) or ubiquitinated Ubc6 (250 nM) were incubated with CPY* (50 nM). After washing, an aliquot of beads were incubated with SDS sample buffer to determine the total bound CPY*. Usp2 (3 µM) was added to the beads and the supernatant was collected. The Usp2 treated samples were then eluted with SDS sample buffer. Samples were analyzed by SDS PAGE and fluorescence scanning. In: CPY* input to the beads, Unb: unbound CPY* after incubation with ubiquitinated Hrd1 or Ubc6. b, Quantification (mean ± s.d.) of the fraction of CPY* released upon deubiquitination by Usp2 from three experiments as in a. c, CPY WT (100 nM) was incubated with fluorescently labeled WT Hrd1 or indicated mutants in liposomes (200 nM), and ubiquitination mix with or without ATP. Samples from indicated time points were analyzed by SDS-PAGE and fluorescence scanning. d, Quantification (mean ± s.d.) of three experiments as in c. e, Increasing concentrations of ubiquitinated, bead-immobilized WT Hrd1 or indicated Hrd1 mutants in liposomes were incubated with fluorescently labeled CPY WT (20 nM). The bound fraction was quantified from supernatants. mean ± s.d (n = 3 independent experiments). f, Liposomes containing fluorescently labeled Hrd1 were incubated with ubiquitination mix containing the ubiquitin K48R mutant. Samples were analyzed by SDS PAGE and fluorescence scanning. Shown is a representative image of two independent experiments. g, Bead-immobilized Hrd1 liposomes were incubated with ubiquitination mix containing WT or K48R ubiquitin with or without ATP. Beads were then subsequently incubated with fluorescently labelled CPY* (40 nM). Samples were analyzed by SDS PAGE and fluorescence scanning. h, Quantification (mean ± s.d.) of three experiments as in g. i, Constant-voltage recordings of ubiquitinated Hrd1 KRK mutant at indicated voltages in the absence (left) and presence of CPY* (right). Shown are representative traces of three independent experiments. Source data and unprocessed gels are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Molecular mechanism for Hrd1-dependent retrotranslocation of misfolded proteins from the ER lumen to the cytosol.

A Misfolded substrate binds to the luminal face of Hrd1 (1). Hrd1 auto-ubiquitination opens the retrotranslocation pore (2) which is further expanded by substrate insertion. A high-affinity binding site on the cytoplasmic face of Hrd1 drives initial substrate translocation (3). The substrate is ubiquitinated by Hrd1 on the cytoplasmic side of the membrane and recruits the Cdc48 complex (4). The Cdc48 complex segregates substrate and Hrd1, and extracts the ubiquitinated substrate from the membrane through rounds of ATP hydrolysis. Hrd1 is deubiquitinated by a DUB, closing the retrotranslocation pore (5).

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Vasic, V., Denkert, N., Schmidt, C.C. et al. Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins. Nat Cell Biol 22, 274–281 (2020). https://doi.org/10.1038/s41556-020-0473-4

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