Starved cells eat ribosomes

nature cell biology volume 10 | number 5 | mAY 2008 505

N e w s A N d v i e w s

lurcher, SCA-1, pcd)14. To date, clear beneficial effects of heterotypic cell fusion of BMDCs with other cells types have only been demon-strated in a mouse model of a lethal liver dis-ease in which the enzyme fumarylacetoacetate hydrolase is absent. In this model, large num-bers of wild-type BMDCs fused with mutant liver cells (presumably under high selective pressure) corrected the metabolic deficiency and rescued the disease phenotype11.

Mechanistically, it is important to note that lipid bilayer membranes do not spontaneously fuse, and that fusion between membranes involves a highly intricate choreography of lipids and proteins, best studied in presyn-aptic neurotransmitter release15. Alterations in the cell membrane that are likely to occur during chronic inflammation and the con-comitant production of a variety of cytokines

may predispose certain cells to fusion events. As Purkinje neurons and cardiomyocytes are difficult to grow in vitro, co-cultures of myelo-lymphoid cells with muscle or liver cells in the presence of specific inflammatory cytokines will be helpful in designing in vitro models of heterotypic cell fusion.

The findings by Johansson et al. and Nygren et al. underscore the importance of learning more about the biological function of heterotypic cell fusion. Clearly, a better understanding of the medical consequences of chronic inflammation, of which hetero-typic cell fusion may be an important aspect, is highly relevant to a range of clinical dis-ciplines. Moreover, revisiting the biological role of cell fusion during disease and tis-sue repair may rescue cell fusion from the Rogue’s gallery.

1. Alvarez-Dolado, M., Front. Biosci. 12, 1–12 (2007).2. Cowan, C. A., Atienza J., Melton D. A., Eggan, K.

Science 309, 1369–1373 (2005).3. Silva, J., Chambers I., Pollard, S., Smith A. Nature 441,

997–1001 (2006).4. Johansson, C. B. et al. Nature Cell Biol. 10, 575–583

(2008).5. Nygren, J. M. et al. Nature Cell Biol. 10, 584–592

(2008).6. Bjornson, C. R., Rietze, R. L., Reynolds, B. A.,

Magli, M. C., Vescovi, A. L. Science 283, 534–537 (1999).

7. Mezey, E., Chandross, K. J., Harta, G., Maki, R. A., McKercher, S. R. Science 290, 1779–1782 (2000).

8. Brazelton, T. R., Rossi, F. M., Keshet, G. I., Blau, H. M. Science 290, 1775–1779 (2000).

9. Ying, Q. L., Nichols, J., Evans, E. P., Smith, A. G. Nature 416, 545–548 (2002).

10. Terada, N. et al. Nature 416, 542–545 (2002).11. Wang, X. et al. Nature 422, 897–901 (2003).12. Alvarez-Dolado, M. et al. Nature 425, 968–973

(2003).13. Weimann, J. M., Johansson, C. B., Trejo, A., Blau,

H. M. Nature Cell Biol. 5, 959–966 (2003).14. Dusart, I., Guenet, J. L., Sotelo, C. Cerebellum 5:163–

173 (2006).15. Jahn, R., Lang, T., Südhof, T. C. Cell 112, 519–533

(2003).

starved cells eat ribosomesHitoshi Nakatogawa and Yoshinori Ohsumi

autophagy is a process in which cytoplasmic components are broken down to supply materials for the synthesis of essential molecules under nutrient-limiting conditions. Because this process involves random sequestration of the cytoplasm by large membrane vesicles, considerable amounts of molecules, such as ribosomes, are necessarily degraded by autophagy. However, starving cells also promote additional selective degradation of ribosomes as a requirement for survival.

Eukaryotic cells are equipped with elabo-rate mechanisms by which they can survive when starved of nutrients. Autophagy (or macroautophagy), a primary pathway for degradation of the cytoplasm in lysosomes or vacuoles1,2, is induced by nutritional dep-rivation, and is essential for adaptation of cells to such environmental changes. When autophagy is induced, a small cup-shaped vesicle called the isolation membrane appears in the cytoplasm, which then expands to form a spherical shape, and closes to form a double membrane-bound vesicle called the autophagosome (Fig. 1). Consequently, the autophagosome randomly encloses a portion of the cytoplasm and often even organelles. Then, the outer membrane of the autophago-some fuses with lysosomes/vacuoles to allow degradation of the contents, together with

the inner membrane, by hydrolases within these lytic compartments. Given that cyto-plasm sequestration occurs in a non-selective manner, and that degradation is catalysed by comparatively non-specific hydrolases, including proteases (peptidases), nucleases, lipases and glycosidases in lysosomes/vacu-oles, autophagy could be involved in turnover of almost all biomolecules. Amino acids pro-duced by autophagy are essential for synthesis of proteins under conditions of starvation3.

Non-selective sequestration means that absolute amounts of molecules degraded by autophagy directly reflect their abundance in the cytoplasm if they are evenly dispersed. Thus, highly abundant molecules and com-plexes such as ribosomes, which occupy about half of cellular proteins and ribonucleic acids, are necessarily subjected to autophagic degra-dation on a large scale. This seems reasonable in light of the cellular strategy whereby superflu-ous constituents are recycled to produce those essential for survival. This would also attenuate

general protein synthesis in the cell. Electron microscopic images of Saccharomyces cer-evisiae cells vigorously undergoing autophagy clearly show that autophagic bodies — inner structures of autophagosomes released into the vacuolar lumen when they fuse with the vacuole — enclose highly electron-dense dots representing ribosomes, in a similar pattern to that in the cytoplasm (Fig. 1; ref. 4); thus, con-siderable amounts of ribosomes seem to be degraded in the vacuole through a non-selec-tive autophagic pathway. In this issue, however, Kraft et al.5 suggest that this alone is insuffi-cient to maintain nutrient-starved yeast cells, which also require further selective autophagic breakdown of ribosomes.

Kraft et al. observed that several green fluo-rescent protein (GFP)-fused ribosomal proteins of both 60S and 40S subunits, which were func-tional and correctly assembled into ribosomes, were delivered to and degraded in the vacuole of nitrogen-deprived yeast cells in an autophagy-dependent manner5. This is not surprising, given

Hitoshi Nakatogawa and Yoshinori Ohsumi are in the Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. e-mail: [email protected]

© 2008 Nature Publishing Group

mailto:[email protected]

506 nature cell biology volume 10 | number 5 | mAY 2008


that autophagy non-selectively delivers cytoplas-mic constituents to the vacuole. However, when the authors compared autophagic degradation of ribosomal proteins with that of other cytoplas-mic proteins, they found that ribosomal degra-dation occurred more extensively. This suggests that ribosomes are preferentially targeted to the autophagic pathway under conditions of nitro-gen starvation. This type of autophagy is called ‘selective’ autophagy, and recently, different ‘car-gos’ have been reported1,2.

By screening starvation-sensitive S. cer-evisiae mutants, Kraft et al. identified the ubiquitin deconjugation enzyme Ubp3p as an essential factor for selective autophagy of ribosomes. In cells lacking this protein, ribos-omal proteins were degraded only as efficiently as control cytoplasmic proteins. Importantly, the efficiency of control protein degradation was not affected by deletion of UBP3. This and other experiments suggest that Ubp3p is not required for starvation-induced, non-selective

autophagy. Similar results were obtained for cells in which BRE5, which encodes a cofac-tor of Ubp3p, had been deleted. In addition, the authors presented data suggesting that other intracellular membrane traffic, includ-ing endocytotic, exocytotic and the multi-ve-sicular-body pathways, are normal in ubp3∆ cells. These results suggest that Ubp3p/Bre5p is specifically involved in selective incorpora-tion of ribosomes into autophagosomes under starvation conditions.

Attenuation of translation during starvation causes the disassembly of polysomes, accom-panied by a concomitant increase of free 60S ribosomal subunits. In ubp3∆ cells exposed to nitrogen starvation, polysome disassembly occurred normally, but, compared with wild-type cells, larger amounts of 60S subunits accumulated, suggesting that Ubp3p-mediated autophagic degradation of ribosomes targets whole 60S subunits rather than being limited to specific ribosomal proteins. In contrast,

the authors showed that degradation of 40S subunits was not affected by UBP3 deletion, although ribosomal proteins constituting 40S subunits were also degraded by autophagy more efficiently than other cytoplasmic proteins in starved cells. An as yet uncharacterized mecha-nism may therefore be responsible for selective autophagy of ribosomal 40S subunits.

How does Ubp3p/Bre5p mediate selective autophagic degradation of ribosomal 60S subunits? The authors showed that if one of the ribosomal proteins was immunoprecipi-tated, several ubiquitinated proteins, likely to be ribosomal or ribosomal-associated pro-teins, were co-precipitated in the ubp3∆ strain to a greater extent than the wild-type strain. Given this and the fact that Ubp3p belongs to the family of ubiquitin deconjugation enzymes that remove ubiquitin from target proteins, the authors discuss the possibility that de-ubiquitination of a ribosomal component(s) or an unknown ‘receptor’ by Ubp3p/Bre5p is required for selective engulfment of 60S subunits by autophagosomes. Finally, Kraft et al. showed that both ubp3∆ and bre5∆ strains are sensitive to nitrogen starvation, supporting the idea that selective autophagic degradation of ribosomes (60S subunits) is important for maintaining cell viability during starvation.

The finding that ribosomes, abundant cyto-plasmic components, are selectively targeted to autophagy even under conditions where simul-taneous bulk degradation of the cytoplasm occurs, is unexpected. Why do starved cells need such extensive degradation of ribosomes? The authors argue that selective autophagy of ribosomes is required for rapid shutdown of protein synthesis, as well as for supply of their degradation products, to maintain cellular homeostasis. However, the extent of selective autophagy of ribosomes, compared with non-selective autophagy, has not been estimated. In addition, starvation-sensitive growth caused by deletions of UBP3 and BRE5 may not be fully attributable to deficient degradation of ribosomal 60S subunits; these proteins may function in a different pathway(s) required for adaptation to starvation. Indeed, previ-ous studies have shown that Ubp3p is also involved in other cellular processes, including degradation of aberrant proteins, membrane traffic from the endoplasmic reticulum to the Golgi apparatus and transcriptional silenc-ing. Therefore, further analysis is required to understand the physiological role(s) of selec-tive autophagy of ribosomes.

AP

IM

V

AB

???

Selective (Ubp3p/Bre5p)

Non-selective

???

Degradation

Figure 1 Schematic diagram of autophagic degradation of ribosomes in yeast. When yeast cells are exposed to nutritional stress, they induce autophagy, by which ribosomes (shown as blue dots) are non-selectively delivered to and degraded in the vacuole, together with other cytoplasmic constituents. In addition, ribosomes are selectively incorporated into autophagosomes; however, this has not been morphologically examined. Ubp3/Bre5 is specifically involved in the uptake of ribosomal 60S subunits into autophagosomes. IM, isolation membrane; AP, autophagosome; AB, autophagic body; V, vacuole.


nature cell biology volume 10 | number 5 | mAY 2008 507


In this study, a relationship between the ubiquitin system and selective autophagy of ribosomes has been suggested. However, the targets of ubiquitination and enzymes involved remain unknown. In addition, how ubiquiti-nation and de-ubiquitination regulates selec-tive uptake of ribosomes by autophagosomes still remains a mystery. Recent studies have revealed that different molecules or com-plexes are selectively recognized and deliv-ered to lysosomes or vacuoles by autophagy1,2. Mechanisms of such cargo selection have been well studied for aminopeptidase I (Ape1p) in yeast, as well as aberrant protein inclusions in mammalian cells. Ape1p is selectively incor-porated into small, autophagosome-like vesi-cles and transported into the vacuole6,7. In this pathway, Ape1p self-assembles and forms an aggregate-like structure. This further interacts with the receptor protein Atg19p (ref. 8), which

is recognized by a vesicle-forming machinery common to that which mediates autophago-some formation. Similarly, in mammalian cells, ubiquitin-positive protein inclusions, relevant to various degenerative diseases, are formed in a p62/sequestosome dependent manner, which are then cleared by autophagy9,10. In this process p62/sequestosome, like Atg19p, also serves as a receptor protein that links those inclusions to an autophagosome-form-ing machinery. In both cases, the assembly of cargo, which can be observed as dots under a fluorescent microscope, is a prerequisite for their efficient engulfment by autophagosomes. On the other hand, we have reported that the acetaldehyde dehydrogenase Ald6p is prefer-entially degraded by autophagy in nitrogen-starved yeast cells11, as shown by Kraft et al. for ribosomes5. Although the dot formation of Ald6p and ribosomes was not observed with

fluorescent microscopic analyses, it is inter-esting to note here that autophagic bodies with contents of higher density than the cyto-plasm are occasionally observed with electron microscopy4. Addressing how these apparently diverse cargos are efficiently incorporated into autophagosomes will shed light on a new type of ‘unbalanced self-eating’.

1. Mizushima, N. Genes Dev. 21, 2861–2873 (2007).2. Xie, Z. & Klionsky, D. J. Nature Cell Biol. 9, 1102–

1109 (2007).3. Onodera, J. & Ohsumi, Y. J. Biol. Chem. 280, 31582–

31586 (2005).4. Takeshige, K. et al. J. Cell Biol. 119, 301–311

(1992).5. Kraft, C. et al. Nature Cell Biol. 10, 602-610 (2008).6. Scott, S. V., Baba, M., Ohsumi, Y. & Klionsky, D. J. J.

Cell Biol. 138, 37–44 (1997).7. Baba, M. et al. J. Cell Biol. 139, 1687–1695

(1997).8. Scott, S. V. et al. Mol. Cell 7, 1131–1141 (2001).9. Bjorkoy, G. et al. J. Cell Biol. 171, 603–614 (2005).10. Komatsu, M. et al. Cell 131, 1149–1163 (2007).11. Onodera, J. & Ohsumi, Y. J. Biol. Chem. 279, 16071–

16076 (2004).

Marked for deathKevin Petrie and Arthur Zelent

sUMOylation of PML–RaRα oncoprotein has been linked to its arsenic-induced degradation and the therapeutic response in acute promyelocytic leukaemia. Two groups identify PML as an in vivo target of the RinG finger ubiquitin e3 ligase RnF4, which specifically binds polysUMOylated PML and is essential for the arsenic-induced catabolism of both PML and PML–RaRα.

Although better known as a poison and famously rumoured to have been used to kill Napoleon, arsenic is one of the oldest medi-cines known to man. First described nearly 2500 years ago by the ancient Greek physician Hippocrates, it has been used to treat a vari-ety of ailments and diseases including syphilis and cancer (for a review see ref. 1). The use of arsenic declined during the twentieth century due to concerns about its toxicity and advances in modern medicine. However, in 1992 it was reported that Ai-ling 1, a Chinese traditional medicine containing high levels of this semi-metal induced dramatic remissions in patients suffering from acute promyelocytic leukaemia (APL). Arsenic trioxide (ATO, As2O3) was iden-tified as the active component and the finding

that it was therapeutically effective in APL but not in other subtypes of acute myeloid leukae-mia suggested that it targeted the PML–RARα fusion oncoprotein, which exerts a dominant-negative effect on both retinoic acid and PML signalling during APL.

Although the specificity of ATO for PML–RARα-associated APL is clear, how it acts has remained contentious. ATO exerts a number of different effects that are to a degree concen-tration-dependent, inducing apoptosis at high concentrations and promoting differentiation at lower levels. Documented effects of ATO include interference with interactions between the SMRT co-repressor and nuclear receptors and induction of reactive oxygen species1. These activities could potentially contribute to the therapeutic effect of ATO in APL; how-ever, it has been demonstrated that both PML–RARα and wild-type PML (but not wild-type RARα) are degraded in APL cells after ATO

treatment. This supports the notion that the PML moiety is the primary target2. Subsequent studies in APL cells showed that ATO treat-ment leads to both PML and PML–RARα becoming highly modified by SUMO proteins, which are related structurally to ubiquitin, and that PML sumoylation can take place at Lys 65, 160 and 490 (ref. 3). A previous study suggested that only Lys 160 SUMOylation is important for ATO-induced degradation of PML and PML–RARα, as this modification also mediates recruitment of the proteasome to PML nuclear bodies4. In this issue of Nature Cell Biology, Lallemand-Breitenbach et al5 and Tatham et al6 demonstrate that the RING finger ubiqui-tin E3 ligase RNF4/SNURF is critical for the SUMOylation-dependent ubiquitination and subsequent catabolism of PML. For PML to be ubiquitinated by RNF4, it must be modified by a polySUMO chain on Lys 160, which is specifi-cally targeted by RNF4 through four tandemly

Kevin Petrie and Arthur Zelent are in the Section of Haemato-Oncology, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. e-mail: [email protected]


mailto:[email protected]

Documents

Starved cells eat ribosomes