Ribophagy Relies on Rpl12

Abstract

The selective decay of ribosomal subunits via autophagy (i.e., ribophagy) is crucial for maintaining cellular protein homeostasis under stress. Findings across various model systems highlight Rpl12’s conserved function in ribophagy and demonstrate its importance in cellular health and the response to environmental stresses.

Ribosomes are complex machines that decode mRNAs to produce proteins. In addition, they are required for mRNA surveillance. In yeast, ribosomes are comprised of 79 ribosomal proteins assembled onto 4 rRNAs¹. Their biosynthesis accounts for about half of all transcription and translation events. Thus, a significant amount of biomass is stored in ribosomes². Keeping these machines in good working order, by repairing or destroying damaged parts, is crucial for maintaining protein homeostasis and cellular health.

Autophagy is a cellular pathway responsible for recycling cellular components, including ribosomes. Autophagy depends on the engulfment of cargoes within vesicles, which then fuse with the lysosome (or vacuole in yeast), leading to the degradation of the cargo to recycle the amino acids and nucleotides. While non-selective autophagy is thought to provide important cellular building blocks during starvation, selective autophagy is important for the degradation of damaged cellular components, including ribosomes. In addition, by destroying pathogens, autophagy is an essential part of the immune response³.

Ribophagy, the selective degradation of ribosomal subunits, has been discovered nearly 20 years ago⁴. And while its contribution to ribosome turnover during nutrient stress might be more limited than originally thought⁵, ribophagy becomes a dominant mechanism for ribosome turnover during proteotoxic stress⁶. However, how ribosomes are selected by the autophagy machinery for targeted degradation, remained unclear. In this issue of Nature Cell Biology, Chen, Hu, Zhao, Fang et al. address this critical question in a tour-de-force study, which revealed a conserved role for the 60S ribosomal protein Rpl12 in ribophagy from yeast to mammals⁷.

Taking advantage of the large body of knowledge of selective autophagy from other systems, the researchers used a pulldown to screen for ribosomal interaction partners of Atg8, which recruits the phagocytic membrane⁸, after nitrogen starvation as well as Atg11. They identified Rpl12, which forms the base of the P-stalk and is accessible on the surface of the 80S ribosome⁹. Critically, they confirmed the interaction of Rpl12 with Atg11, which occurs via a binding surface important for selective autophagy of other cargoes. Moreover, they identified (and then mutated) the Rpl12 residues that bind to Atg8, providing for a valuable reagent, which they use to demonstrate that the Atg8/Rpl12 interaction is required for ribophagy (Figure 1).

Figure 1. Schematic overview of the mechanism of ribophagy mediated by Rpl12, a conserved autophagy receptor:

Rpl12, as part of the 60S subunit, is phosphorylated by Atg1 (denoted by the p), which strengthens its interaction with Atg11, leading to the formation of a PAS (phagosome attachment site). In addition, Rpl12 binds Atg8, which recruits the membrane to engulf the ribosome.

Extending their mechanistic studies, Chen, Hu, Zhao, Fang et al. next demonstrated that the Atg1 kinase, a key regulator of autophagy⁸, phosphorylates Rpl12, which strengthens Rpl12’s interaction with Atg11, thereby supporting the formation of the phagosome assembly site (PAS) and triggering ribophagy (Figure 1). Again, careful identification and validation of the phosphorylation sites enabled the generation of a valuable reagent to demonstrate the importance of Atg1-mediated phosphorylation of Rpl12. Overall, using comprehensive biochemical and mass spectrometry-based assays, Chen, Hu, Zhao, Fang et al. showed that when Rpl12 is not functioning properly, there is a buildup of ribosomal proteins and rRNAs that are not efficiently degraded, making it difficult to maintain protein homeostasis in the cell.

In order to investigate the role of Rpl12 as a ubiquitous ribophagy receptor, the authors generated homologous Rpl12 functional mutants in C. elegans, Drosophila, human cells and a mouse model, and went on to show that the role of Rpl12 as an autophagy adaptor is indeed conserved, likely using a conserved mechanism, as the Atg8 binding-deficient mutant was defective in ribophagy in all cases.

Importantly, the use of these varied model systems, as well as the point mutant that Chen, Hu, Zhao, Fang et al. generated, allowed them to provide important insights into the role of ribophagy for such varied physiological conditions as survival during starvation (in yeast, worms, flies, mammals), pathogenic infection (worms, flies), development (worms, flies), aging (worms, flies), and liver glucose metabolism (mouse). Altogether, the broad experiments conducted in yeast, nematodes, flies and mammals provide convincing evidence that Rpl12 is a conserved autophagy receptor, which helps to form a PAS and recruits the phagosome membrane. Even more excitingly, the authors set the stage for deeper studies of the physiological roles of ribophagy from yeast to humans.

Future studies will likely take advantage of the autophagy-deficient ribosomes generated here to further decipher the role of ribophagy in cellular health and disease. For example, previous work has indicated that autophagy is important in cancer prevention, but is also activated during cancer development¹⁰. Whether this involves ribosomes during at least one stage will be exciting to decipher. Similarly, the role of ribophagy for survival during starvation and in life-span extension appears paradoxical, as starvation increases lifespan, thus meriting further scrutiny.

Moreover, it remains unknown whether all ribosomes are targeted via Rpl12-mediated ribophagy, or just the damaged ones. Additionally, how 60S subunits are distinguished from 80S complexes to enable their selective degradation (leaving behind 40S subunits) is not well delineated in the study, as Rpl12 is accessible in 80S complexes.

Finally, the research also raises interesting questions about whether this recycling process works the same way in all cell types. For example, the authors found that ribophagy-defective Rpl12 affects liver metabolism in the mouse model, and Rpl12 fly mutants displayed exacerbated muscle degeneration and impairment in motor functions. The cell-type specific nature suggests that different cells might use ribophagy in different ways. Indeed, in yeast, Rpl12 is not essential, demonstrating that Rpl12-deficient ribosomes are at least partially functional. Moreover, overexpression of Rpl12 promoted ribophagy, implying that not all ribosomes have Rpl12. Indeed, previous work has shown high levels of Rpl12 in the spleen and low levels in fat cells¹¹, perhaps indicating a different role for ribophagy in these tissues, which is worth further exploring.

In summary, by identifying a receptor on 60S ribosomes, this work offers a deeper understanding of how cells maintain ribosome homeostasis via the selective degradation of ribosomes, provides important insights into the role of ribophagy in cellular physiology, and enables future studies into both the mechanism of ribophagy and its physiological and tissue-specific roles.