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Receptor oligomerization guides pathway choice between proteasomal and autophagic degradation

Nature Cell Biology volume 19, pages 732–739 (2017)Cite this article

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Abstract

Abnormal or aggregated proteins have a strong cytotoxic potential and are causative for human disorders such as Alzheimer’s, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis1,2,3. If not restored by molecular chaperones, abnormal proteins are typically degraded by proteasomes or eliminated by selective autophagy1,2,3,4,5. The discovery that both pathways are initiated by substrate ubiquitylation but utilize different ubiquitin receptors incited a debate over how pathway choice is achieved2,6,7,8,9,10,11,12,13. Here, we demonstrate in yeast that pathway choice is made after substrate ubiquitylation by competing ubiquitin receptors harbouring either proteasome- or autophagy-related protein 8 (Atg8/LC3)-binding modules. Proteasome pathway receptors bind ubiquitin moieties more efficiently, but autophagy receptors gain the upper hand following substrate aggregation and receptor bundling. Indeed, by using sets of modular artificial receptors harbouring identical ubiquitin-binding modules we found that proteasome/autophagy pathway choice is independent of the ubiquitin-binding properties of the receptors but largely determined by their oligomerization potentials. Our work thus suggests that proteasomal degradation and selective autophagy are two branches of an adaptive protein quality control pathway, which uses substrate ubiquitylation as a shared degradation signal.

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Figure 1: Substrate specificity of proteasome pathway Dsk2 and autophagy pathway Cue5 ubiquitin receptors.
Figure 2: Role of ubiquitylation enzymes and ubiquitin chain types in substrate targeting by Dsk2 and Cue5.
Figure 3: Substrate binding properties of Dsk2 and Cue5 ubiquitin receptors.
Figure 4: Cue5 forms oligomers involving its CUE domain.
Figure 5: Synthetic receptors reveal requirements for proteasome/autophagy pathway choice.

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Acknowledgements

We thank B. Schulman, B. Pfander and L. Cairo for comments on the manuscript, I. Psakhye for early contributions and discussions, A. Strasser for technical assistance, N. Nagaraj and S. Uebel for mass spectrometry, protein analysis and isothermal titration calorimetry assays, S. Chanarat for Cue5-CUE structure modelling and S. Lindquist (Whitehead Institute for Biomedical Research, USA) and F.-U. Hartl (Max Planck Institute for Biochemistry, Germany) for materials. S.J. is supported by the Max Planck Society, Deutsche Forschungsgemeinschaft, Centre for Integrated Protein Science Munich, Louis-Jeantet Foundation, and a European Research Council (ERC) Advanced Grant.

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  1. Kefeng Lu

    Present address: State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and National Collaborative Innovation Center for Biotherapy, 610041, Chengdu, China

  2. Stefan Jentsch: Deceased 29 October 2016.

Authors and Affiliations

  1. Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany

    Kefeng Lu, Fabian den Brave & Stefan Jentsch

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  1. Kefeng Lu
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Contributions

K.L. conducted and designed the experiments, F.d.B. and S.J. contributed to the experimental designs, all authors interpreted the data and prepared the paper, and S.J. wrote the manuscript.

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Correspondence to Kefeng Lu or Fabian den Brave.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Substrate specificity of ubiquitin receptors.

a, Degradation of Ub-βgal-WT in WT and mutant cells. Degradation is strongly impaired in cells deficient in Dsk2, mildly impaired in cells lacking the Dsk2-related protein Rad23, but efficiently degraded in cells lacking the autophagy proteins Cue5 or Atg8. Experiment was performed as in Fig. 1e. Representative image of two independent replicates is shown. b, Cue5 binds the aggregation-prone substrate variant Ub-βgal-X90 stronger than Ub-βgal-WT. Binding does not occur when the ubiquitin-binding CUE is altered (CUEmut). Experiment was performed as in Fig. 3a. Representative image of two independent replicates is shown. c, In contrast to Dsk2-GFP, Cue5-GFP co-localized extensively with TDP-43-mars aggregates (scale bar, 5 μm). Wide-field of cells similar to Fig. 1i. Representative image of two independent replicates is shown. Unprocessed original scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 2 Ubiquitin modification specificity of Dsk2 and Cue5.

a, ITC analysis of binding properties of the UBA domain of Dsk2 and the CUE domain of Cue5 to mono-ubiquitin and K48- or K63-linked di-ubiquitin. Exceptional injections were excluded. Representative image of two independent replicates is shown. b, Dsk2 with UBA domain replaced by the CUE domain of Cue5 (Dsk2-CUE) binds endogenous ubiquitin conjugates with lower efficiency. Representative image of two independent replicates is shown. c, Dsk2-CUE rescues viability of dsk2 and rad23 double deficient cells at 37 °C. Representative image of two independent replicates is shown. Unprocessed original scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 3 Cue5 forms oligomers involving its CUE domain.

a, Different regions of Cue5 were purified and subjected to cross-linking as in Fig. 4b. Representative image of two independent replicates is shown. b, Alignment of CUE domain sequences from Cue5, Cue2 and Vps9. The conserved amino acids important for dimerization are boxed. Residues L135-L136 and F109-P110 (in red) are shown in modelled structure of Cue5 CUE domain using UCSF chimera based on Vps9 CUE domain (PDB ID code 1P3Q). Purple: ubiquitin, light blue: CUE (monomer), light green: CUE (monomer). c, Amino acid replacements within the CUE domain of Cue5 impair self-interaction. GST-pull-down assays were performed using His-tagged Cue5 and GST-fusions of WT Cue5 or CUE domain mutant variants of Cue5 (Cue5-F109A, P110A; Cue5-L135A, L136A). A Cue5 variant harbouring amino acid replacements C-terminal of the bona fide CUE domain (Cue5-L174A, L175A) was used as control. Representative image of two independent replicates is shown. d, Amino acid replacements described above impair ubiquitin binding. Experiments were carried out similar as in c with purified proteins and yeast cell lysates. Representative image of two independent replicates is shown. Unprocessed original scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 4 Functionality of synthetic receptors.

a, Expression levels of synthetic receptors. Yeast cells expressing the synthetic receptors described in Fig. 5a were analysed as in Fig. 3d. Representative image of two independent replicates is shown. b, Oligomerization potential of the artificially constructed autophagy receptors shown in Fig. 5a. Constructs were purified at concentration of 1 mg ml−1 and exposed to formaldehyde-cross-linking (1.85%) for 1 h prior to analysis by SDS polyacrylamide gel electrophoresis. Coomassie-stained gel is shown. Representative image of two independent replicates is shown. c, Degradation of aggregation-prone Ub-βgal-X90 (top panel) mediated by the synthetic oligomeric receptor (2FK-OAIM) is independent of Dsk2 (Δdsk2Δcue5 lanes) but depends on the autophagy pathway (Δatg8Δcue5 lanes). By contrast degradation of soluble Ub-βgal-WT by monomeric receptor (2FK-MUBL−AIM) (bottom panel) is independent of autophagy. Experiment was performed as in Fig. 1e. Representative image of two independent replicates is shown. d, The synthetic oligomeric receptor (2FK-OAIM) is as potent as Cue5 in mediating degradation of aggregation-prone Ub-βgal-X90. In contrast, the dimeric receptor (1FK-DAIM) is not able to support efficient degradation of Ub-βgal-X90. Experiment was performed as in Fig. 1e. Representative image of two independent replicates is shown. e, Differential binding of monomeric and oligomeric synthetic receptors to Ub-βgal-WT and Ub-βgal-X90. GST-pull-down assays were performed as in Fig. 1g. Representative image of two independent replicates is shown. f, Binding of monomeric and oligomeric synthetic receptors to the proteasome is similar and does not affect Dsk2 binding to the proteasome. Co-IP assays with the indicated antibodies were performed as in Fig. 3a. Representative image of two independent replicates is shown. g, The oligomeric receptor (2FK-OUBL) shows no dominant negative effect on degradation of Ub-βgal-WT in the presence of Dsk2. The experiment was performed as in Fig. 1e. Representative image of two independent replicates is shown. h, The oligomeric receptor (2FK-OUBL) shows no negative effect on cell growth. The experiment was also performed as in Supplementary Fig. 2c. Representative image of two independent replicates is shown. i, Co-localization of GFP-tagged oligomeric (2FK-OAIM), but not monomeric (2FK-MAIM) receptors with mars-labelled TDP-43 protein aggregates. Dimeric receptors (1FK-DAIM) show only weak co-localization with aggregates (scale bar, 5 μm). Wide-field of cells similar to Fig. 5d. Representative image of two independent replicates is shown. Unprocessed original scans of blots are shown in Supplementary Fig. 5.

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Lu, K., den Brave, F. & Jentsch, S. Receptor oligomerization guides pathway choice between proteasomal and autophagic degradation . Nat Cell Biol 19, 732–739 (2017). https://doi.org/10.1038/ncb3531

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