Finding a ribophagy receptor

ABSTRACT

Although ribophagy was demonstrated in budding yeast a decade ago, a specific receptor for this process has been unknown. Recently, a study revealed that NUFIP1 (nuclear FMR1 interacting protein 1) functions as a receptor for the selective degradation of ribosomes by starvation-induced autophagy in cultured mammalian cells. In addition to the identification of a selective autophagy receptor, this study suggests a strategy that can be adapted to the identification of additional novel receptor proteins.

KEYWORDS:

• Autophagy
• cargo
• receptor
• ribosomes
• selective autophagy

Macroautophagy/autophagy can be categorized as nonselective or selective based on the mechanism of how the cargos are captured by the phagophore, the precursor to the autophagosome. Our understanding of the types of cellular components that can be degraded through autophagy keeps expanding. However, the identification of a new autophagic cargo does not necessarily equate with the discovery of a new type of selective autophagy; cargo can be degraded through nonselective autophagy by random engulfment, as long as its size does not exceed the capacity of the phagophore. A gold standard for defining a selective autophagy can be summarized as the cargo-ligand-receptor-Atg8-family protein model [1–3]. In particular, a receptor is required for the degradation of a specific cargo, but not for general degradation mediated by nonselective autophagy. Therefore, finding a cargo-specific receptor is the key to identifying a new type of selective autophagy. Many such types of selective autophagy and their corresponding cargo-specific receptors have been characterized in different organisms, from yeasts to mammals, including the cytoplasm-to-vacuole targeting pathway, mitophagy, pexophagy, reticulophagy, and xenophagy. Degradation of ribosomes upon starvation has been observed previously, and, in yeast, the selective autophagic degradation of ribosomes is Ubp3 and Bre5 dependent [4]; however, without knowing the actual receptor, the detailed mechanism of ribophagy has remained a puzzle until recently. The group of David Sabatini has identified NUFIP1 as a receptor for starvation-induced ribophagy [5].

To study the regulation of the lysosomal proteome by MTORC1, Wyant et al performed LysoIP, a method the authors developed previously for a rapid purification of lysosomes; they compared lysosomes isolated from HEK 293T cells with or without Torin1 (an MTORC1 inhibitor) treatment. Among the candidate proteins whose enrichment in lysosomes was significantly increased upon MTORC1 inhibition, NUFIP1 drew their attention. NUFIP1’s previously identified roles were mainly localized in the cell nucleus, but it has been reported that NUFIP1 shuttles to the cytoplasm from the nucleus and colocalizes with ribosomes in neurons [6]. To further study the role of NUFIP1 upon MTORC1 inhibition, the authors first manually confirmed the redistribution of NUFIP1 from the nucleus to lysosomes after Torin1 treatment using cell fractionation and fluorescence microscopy. Autophagy is induced when MTOR activity is inhibited, and the authors observed that in addition to lysosomes, NUFIP1 also colocalizes with autophagosomes. Furthermore, the translocation of NUFIP1 to lysosomes is autophagy dependent; NUFIP1 does not localize to lysosomes isolated from ATG7-depleted cells even after Torin1 treatment.

The data suggest that NUFIP1’s accumulation within lysosomes upon MTOR inhibition is due to its potential role in autophagy. Analysis of the NUFIP1 sequence predicts multiple potential LC3-interacting regions (LIRs), and one of them is indeed required for the binding of the protein with LC3B. NUFIP1’s direct interaction with LC3B, and its previously reported colocalization with ribosomes, led to the hypothesis that NUFIP1 is a receptor for ribophagy in mammals. In multiple cell types the authors tested, NUFIP1 depletion prevents autophagic degradation of ribosomal proteins and rRNAs that would normally be caused by MTOR inhibition. Moreover, electron microscopy data suggest that NUFIP1 containing a LIR mutation cannot rescue the reduced engulfment of ribosomes into phagophores that is seen in NUFIP1 null cells. These data strongly support the proposal that NUFIP1 is a selective autophagy receptor of ribosomes upon MTOR inhibition. Therefore, the missing piece of the selection machinery for ribophagy, a receptor, has finally been identified, at least for mammals.

Still, there remains a missing link to complete the cargo-ligand-receptor-Atg8-family protein model of ribophagy. That is, what is the ligand? In other words, which part of the ribosome is recognized by NUFIP1, and how is the interaction regulated by MTORC1? This question was only partially answered in the present study. NUFIP1 comigrates with ribosomes during fractionation of Torin1-treated cell lysates, and when EDTA is applied to disassociate ribosomal subunits, NUFIP1’s comigration with the large (60s) ribosomal subunits increases. Moreover, only ribosomes purified from Torin1-treated cell lysates strongly interact with NUFIP1. These results suggested that part of the large ribosomal subunit in its MTOR-inactive-state can be the ligand for ribophagy, which is recognized by NUFIP1. However, it still remains unknown as to which part of the large ribosomal subunit functions as the actual ligand, and what is the specific nature of the MTOR-regulated modification of this ligand that takes place during ribophagy.

In addition to adding one more member to the growing list of selective autophagy receptors, this study provides some new insights into finding more receptors. Previously, researchers screened for selective autophagy receptors using cargo-centered methods. That is, copurification of proteins with the autophagy cargos, or a genetic screen of the deletion/knockdown libraries looking for decreased degradation of the cargo were commonly used approaches. In budding yeast, many selective autophagy receptors interact with Atg11, the scaffold protein for selective autophagy [1–3]. Therefore, proteins that interact with Atg11 became good candidates for selective autophagy receptors. Unfortunately, no Atg11 homolog has been identified in mammals. Instead, because these receptors interact with LC3B/Atg8, the presence of a LIR motif is a common feature of receptors that is more useful for analyses in more complex eukaryotes. The success of finding a mammalian ribophagy receptor in the study by Wyant et al. illustrates the utility of using another common feature of selective autophagy receptors for identifying additional receptor proteins: In mammalian cells, the final destination of all these receptors, regardless of their recognized cargos, is the lysosome. Receptors for selective autophagy will accumulate in lysosomes under the appropriate stress and treatment conditions. Therefore, using a similar strategy as in this study, by combining LysoIP proteomic analysis of cells under different types of stress combined with a sequence analysis of potential LIRs, we may expect to identify additional receptors for selective autophagy in the near future.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Institute of General Medical Sciences [GM053396].