The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

ABSTRACT

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.

KEYWORDS:

• autophagy
• lysosome
• plants
• stress
• vacuole

In eukaryotes, there are 2 primary pathways for degradation: the ubiquitin-proteasome system and autophagy. Whereas the UPS is responsible for degrading short-lived proteins, autophagy is considered to be principally responsible for the turnover of long-lived proteins, large protein complexes and organelles. The 26S proteasome occupies a central role in the UPS, and consists of 2 subparticles: a 20S core protease (CP) that contains peptidase active sites, and a 19S regulatory particle (RP) that plays a role in identifying appropriate substrates and translocating them into the lumen of the CP for subsequent breakdown.^1,2 The activity of the proteasome is also regulated at different levels, from transcriptional regulation to post-translational modifications (in particular, ubiquitination).^3,4 Autophagy occurs at a basal level but is further upregulated in response to a variety of stimuli. In addition, autophagy can selectively remove different cellular components with the help of specific receptors, which usually have affinity for ATG8.^5,6 Using Arabidopsis proteasomes tagged with GFP, Marshall et al. observed that autophagy is required for the delivery of proteasomes into the plant vacuole.⁷ They further demonstrated that both nitrogen starvation and chemical or genetic inhibition of the proteasome can induce autophagic degradation of the 26S proteasome, through a process termed proteaphagy. Starvation leads to nonselective proteaphagy, whereas proteasome inhibition results in selective proteasome degradation, which requires the proteasome subunit RPN10 as a specific receptor; RPN10 simultaneously interacts with ubiquitinated proteasome subunits and lipidated ATG8 residing on the phagophore.

The authors detected higher amounts of many 26S proteasome subunits in autophagy-defective mutants despite unchanged transcript levels, suggesting that they might normally be degraded via autophagy. To validate this hypothesis, the authors generated Arabidopsis lines in which they replaced PAG1 (a CP subunit) and RPN5a (an RP subunit) with GFP-tagged versions. Under growing conditions, the proteasome is detected in both the nucleus and cytoplasm; however, after starvation and pre-treatment with the vacuolar-type H⁺-ATPase inhibitor concanamycin A, more GFP puncta are found within vacuoles, and these puncta colocalize with mCherry-ATG8a. In contrast, these vacuolar puncta are not detected in autophagy-defective mutants, indicating that they are of autophagic origin. To exclude the possibility that these puncta contain unincorporated proteasome subunits, the authors utilized 2 fluorescent derivatives of the proteasome inhibitor epoxomycin, allowing them to monitor intact proteasomes. Their data supported the conclusion that the entire proteasome, not just individual subunits, are degraded by autophagy.

Subsequently, the authors examined other conditions for proteaphagy induction. Treatment with the proteasome inhibitor MG132 resulted in a substantial increase in proteaphagy. By examining the vacuole-dependent processing of GFP-tagged proteasome subunits, Marshall et al. were able to show that although both nonselective and selective autophagy participate in degrading proteasomes, MG132 treatment only induces the selective pathway. The authors further demonstrated that proteasome inhibition induced ubiquitination of proteasome subunits, and that this modification is part of the proteaphagy mechanism; association of RPN10 with the proteasome increased following treatment with MG132, and decreased when the proteasomes were treated with the deubiquitinase USP2, suggesting that RPN10 binds to ubiquitinated proteasomes.

Because autophagy receptors typically interact with ATG8, the authors next examined whether RPN10 also binds to this protein. An interaction between RPN10 and ATG8 was confirmed using yeast two-hybrid assays, in vitro affinity isolation and bimolecular fluorescence complementation in planta. RPN10 contains 3 ubiquitin-interacting motifs (UIMs) in its C-terminal domain, among which UIM1 is necessary for ubiquitin binding; in this report, they demonstrated that UIM2 is the binding site in RPN10 for ATG8. Thus, RPN10 can bind to both ubiquitin and ATG8 simultaneously, allowing it to act as a specific cargo receptor. To examine this model, the authors utilized an rpn10-1 mutant line lacking the 3 UIMs. Measuring autophagic flux by fluorescence microscopy and processing of PAG1-GFP, the authors found that the rpn10-1 mutant only blocks proteaphagy induced by proteasome inhibition, but does not affect starvation-induced nonselective proteaphagy. Furthermore, affinity isolation demonstrated that His₆-tagged ATG8e could precipitate free poly-ubiquitin chains in the presence of RPN10 only when the latter contained both UIM1 and UIM2, supporting the model that RPN10 may work as a receptor to link ubiquitinated proteasomes to ATG8–PE.

In conclusion, the authors present an attractive working model for proteaphagy in Arabidopsis. Based on their findings, there are 2 distinct proteaphagic routes: One such process is nonselective proteaphagy, which is induced by starvation, and the other involves a selective mechanism in response to proteasome inhibition. Both routes depend on the autophagy core machinery; however, the selective pathway is apparently independent of ATG13 (and presumably ATG1) because it occurs normally in atg13 mutant lines. The induction of selective proteaphagy also correlates with ubiquitination of the proteasome, where the RPN10 subunit functions as a specific receptor due to its ability to bind both ubiquitin and ATG8. RPN10 thus has a dual role, acting to bind ubiquitinated substrates and facilitating their subsequent degradation as part of the RP lid, but also binding to ubiquitinated proteasome subunits to direct degradation of the proteasome via proteaphagy.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH grant GM053396 to DJK.