Proteasome condensate formation is driven by multivalent interactions with shuttle factors and K48-linked ubiquitin chains

Background:

The proteasome complex is a large and intricate cellular structure responsible for protein degradation and turnover. It is responsible for breaking down and recycling proteins that are damaged, misfolded, or no longer needed by the cell and helps to maintain cellular homeostasis.

The proteasome consists of protein subunits arranged in a cylindrical structure with a hollow core. It can be divided into two main components: the regulatory particle (also known as the 19S particle) and the core particle (also known as the 20S particle). These components work together to recognize, unfold, and degrade targeted proteins into smaller peptides in a highly controlled and selective manner. The 19S regulatory particle is responsible for recognizing and binding to ubiquitinated proteins, which have been tagged with multiple ubiquitin molecules as a signal for degradation. The 20S core particle is the proteolytic component of the proteasome complex.

Generally, polyubiquitinated substrates are recognized by intrinsic ubiquitin receptors present on the 19S (Rpn1, Rpn10, and Rpn13). Also,  extrinsic receptors, known as ubiquitin shuttle proteins (Rad23, Dsk2, and Ddi1) interact with ubiquitinated proteins and deliver them to the proteasome for degradation. All the shuttle proteins share common features, such as their ability to bind to the ubiquitinated protein via the UBA domain and their interaction with the proteasome via the UBL domain. Here, the authors of the preprint propose a model for proteasome condensate formation, where shuttle factors play a critical role.

 

What is our current understanding of proteasome storage granule formation?

Laporte et al. demonstrated that in budding yeast, upon entry into a quiescence state, proteasome subunits re-localize from the nucleus to the cytoplasm. Furthermore, these cytoplasmic proteasome reservoirs, known as proteasome storage granules (PSGs) rapidly mobilize upon exit from quiescence. Upon carbon depletion, yeast relocalizes their proteasomes to form PSGs to be protected from autophagic degradation, and the addition of glucose triggers quiescence exit and the disassembly of the PSGs.

Peters et al. showed that shifting yeast cells to anaerobic conditions by uncoupling the mitochondrial oxidative phosphorylation, lowering the cytosolic pH, and dropping cellular ATP levels, resulted in proteasome sequestration into PSGs. Gu et al. showed by using ubi4Δ yeast cells that deleting the ubiquitin gene almost abrogated PSG formation. Furthermore, the study demonstrated that free ubiquitin enrichment within PSGs.

How such proteasome condensates are formed is not very well understood. In the current preprint, the authors propose a model for PSG formation using yeast as a model organism. For the first time, the authors could demonstrate the role of shuttle factors Rad23, Dsk2, and Ddi1 in PSG formation, during which long 48-liked ubiquitin chains play a pivotal role. This study aimed to investigate the alternative role of the shuttle factors in proteasome condensate formation, which is novel in the field of proteasome stress granules.

 

Key findings of the study:

Generally, proteins that have the tendency to undergo liquid–liquid phase separations in an aqueous environment are often intrinsically disordered. Because proteasome subunits are well-folded proteins with only a few intrinsically disordered domains, it is unclear which factors assist proteasomes in forming condensates. The authors reported that an inhibitor of mitochondrial oxidative phosphorylation, sodium azide-induced PSGs formation is Rad23 and Dsk2-dependent, unlike glucose starvation-induced proteasome condensates. Surprisingly, the study could show that the lack of Ddi1 protease activity triggered PSG formation. The authors also demonstrated that specifically reducing K48-linked ubiquitin chains, but not K63-linked chains, abrogated proteasome condensate formation. Furthermore, the current study indicated that PSG formation following glucose starvation is critically dependent on proteasome intrinsic receptor Rpn10, Whereas sodium azide-induced condensate formation is dependent on Rpn13.

 

Figure 1

Adopted from Figure 1 of Waite et al. (2023), BioRxiv. A) Domain organization of shuttle factors. B) Yeast cells were grown logarithmically, then treated with sodium azide for 24 or 48 hours and condensate formation is dependent on Rad23 and Dsk2. C) Yeast cells were starved for glucose for 24 hours and condensate formation is independent of Rad23 and Dsk2. Adopted from Figure 4 of Waite et al. (2023), BioRxiv. D) Yeast cells were grown in YPD media and observed proteasome condensates in wild-type and dead Ddi1^D220N mutant.

 

Figure 2

Adopted from Figure 4 of Waite et al. (2023), BioRxiv. A) Overexpression of the K48R ubiquitin mutant significantly reduced the percentage of proteasome condensates. Adopted from Figure 5 of Waite et al. (2023), BioRxiv. B) Glucose starvation-induced and azide-induced PSG formation abrogated by Rpn10 mutation and Rpn13 deletion, respectively.

 

Questions for the authors:

• Unlike inhibition of mitochondrial oxidative phosphorylation, Glucose starvation did not block PSG formation in rad23Δ dsk2Δ cells. This suggests that inhibition of mitochondrial oxidative phosphorylation or glucose starvation-induced PSG formation is regulated by alternative factors or pathways. Any thoughts on this?
• Rad23 K7A, I45A, and L31A mutants were shown to significantly abrogated proteasome interaction. As Rad23, Dsk2 is known to interact with proteasome using the UBL domain, whether abrogating proteasome interaction has any effect on proteasome condensate formation.
• Overexpression of lysine-less ubiquitin is shown to induce premature PSG formation in yeast. On the contrary, the authors here observed that long K48-linked ubiquitin chains are critical components of proteasome condensates. Did the authors consider performing a colocalization experiment in the presence of the K48-only ubiquitin variant (allowed only K48-linkage formation), to check whether PSG colocalizes with the K48-linked ubiquitin chain?
• Study demonstrated that the protease dead mutant of Ddi1 (Ddi1^D220N) did not inhibit proteasome condensate formation, indicating the stabilization of longer poly-ubiquitinated substrates as a critical factor for PSG formation. As proteasome inhibition and chain-specific deubiquitinase (DUB) deletion could also similarly stabilize the longer poly-ubiquitinated substrates, did the authors check whether proteasome inhibition or 48-linkage-specific DUB deletion could trigger PSG formation?

 

References

Laporte D, Salin B, Daignan-Fornier B, Sagot I. Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J Cell Biol. 2008 Jun 2;181(5):737-45. doi: 10.1083/jcb.200711154.

Laporte D, Lebaudy A, Sahin A, Pinson B, Ceschin J, Daignan-Fornier B, Sagot I. Metabolic status rather than cell cycle signals control quiescence entry and exit. J Cell Biol. 2011 Mar 21;192(6):949-57. doi: 10.1083/jcb.201009028.

Martinez-Fonts K, Davis C, Tomita T, Elsasser S, Nager AR, Shi Y, Finley D, Matouschek A. The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates. Nat Commun. 2020 Jan 24;11(1):477. doi: 10.1038/s41467-019-13906-8.

Shi Y, Chen X, Elsasser S, Stocks BB, Tian G, Lee BH, Shi Y, Zhang N, de Poot SA, Tuebing F, Sun S, Vannoy J, Tarasov SG, Engen JR, Finley D, Walters KJ. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science. 2016 Feb 19;351(6275):10.1126/science.aad9421 aad9421. doi: 10.1126/science.aad9421.

Peters LZ, Hazan R, Breker M, Schuldiner M, Ben-Aroya S. Formation and dissociation of proteasome storage granules are regulated by cytosolic pH. J Cell Biol. 2013 May 27;201(5):663-71. doi: 10.1083/jcb.201211146. Epub 2013 May 20.

Enenkel C, Kang RW, Wilfling F, Ernst OP. Intracellular localization of the proteasome in response to stress conditions. J Biol Chem. 2022 Jul;298(7):102083. doi: 10.1016/j.jbc.2022.102083. Epub 2022 May 27.

Gu ZC, Wu E, Sailer C, Jando J, Styles E, Eisenkolb I, Kuschel M, Bitschar K, Wang X, Huang L, Vissa A, Yip CM, Yedidi RS, Friesen H, Enenkel C. Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast. Mol Biol Cell. 2017 Sep 15;28(19):2479-2491. doi: 10.1091/mbc.E17-03-0162. Epub 2017 Aug 2.

Tags: proteasome, proteasome storage granule, shuttle factors, yeast

doi: https://doi.org/10.1242/prelights.35477

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