Lipophagy: Selective Catabolism Designed for Lipids

Developmental Cell

Previews of ping-pong is played by Aub and Ago3 in the nuage. To gain insight into the function of piRNA pathway components, Malone et al. (2009) examined the effects of mutating various genes previously implicated in the piRNA pathway, as well as mutation of the flamenco locus. Four of the genes are essential for localization of Ago3 to the nuage—Aub, krimper, spindle-E, and vasa. Ago3, conversely, is required for Aub’s placement in the nuage. Interestingly, previous work showed interdependence among Maelstrom (a putative piRNA pathway component), Aub, and spindle-E for nuage localization, suggesting that nuage may be dedicated to RNA processing via short RNA mechanisms (Findley et al., 2003). Three genes, in addition to Ago3 and Aub, were found to play a role in ping-pong amplification: spindle-E, krimper, and zucchini. The flamenco locus mutation did not affect piRNAs that exhibit a ping-pong signature, confirming the Ago3 and Aub independent function of somatic piRNA biogenesis. The combination of mutational analysis and deep sequencing in these papers thus provides evidence for three subgroups of piRNAs: two in the germline, produced

by Ago3/Aub dependent ping-pong amplification, and one in the soma, produced without the ping-pong amplification step. Group I is predominantly antisense, group II is predominantly sense, and the somatic group III is antisense and independent of Ago3 and Aub. Important questions about piRNAs remain unanswered. First, it is unclear whether we can generalize from flies to mammals. Although PIWI-related proteins are found in mammals, piRNA loci are devoid of transposons, and the structure of mammalian repetitive elements is different from that in flies. Sequences producing piRNAs in mammals are reported to be the fastest evolving loci in the mammalian genome, suggesting that powerful selection is acting on these loci (Assis and Kondrashov, 2009). We also do not know how piRNAs are directed to specific PIWI family proteins. Nor do we understand whether transcription of the antisense strand, on which the pingpong model depends, is constitutive, stochastic, or actively regulated. Finally, we still do not know how piRNA suppress transposons: does the degradation of transposon transcripts prevent their proliferation, or do piRNAs specify silencing by

chromatin modifications, or both? We anticipate surprises.

perhaps

REFERENCES Assis, R., and Kondrashov, A.S. (2009). Proc. Natl. Acad. Sci. USA, in press. Published online April 8, 2009. 10.1073/pnas.0900523106. Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R., and Hannon, G.J. (2007). Cell 128, 1089–1103. Brennecke, J., Malone, C.D., Aravin, A.A., Sachidanandam, R., Stark, A., and Hannon, G.J. (2008). Science 322, 1387–1392. Findley, S.D., Tamanaha, M., Clegg, N.J., and Ruohola-Baker, H. (2003). Development 130, 859–871. Ghildiyal, M., and Zamore, P.D. (2009). Nat. Rev. Genet. 10, 94–108. Klattenhoff, C., Bratu, D.P., McGinnis-Schultz, N., Koppetsch, B.S., Cook, H.A., and Theurkauf, W.E. (2007). Dev. Cell 12, 45–55. Li, C., Vagin, V.V., Lee, S., Xu, J., Ma, S., Xi, H., Seitz, H., Horwich, M.D., Syrzycka, M., Honda, B.M., et al. (2009). Cell 137, 509–521. Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam, R., and Hannon, G.J. (2009). Cell 137, 522–535. Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., and Zamore, P.D. (2006). Science 313, 320–324.

Lipophagy: Selective Catabolism Designed for Lipids Hilla Weidberg,1 Elena Shvets,1 and Zvulun Elazar1,* 1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.05.001

Until recently, degradation of lipid droplets (LDs) has been thought to take place in the cytosol by resident lipases. In a recent issue of Nature, Singh and coworkers describe the involvement of selective autophagy in the delivery of lipid droplets for lysosomal degradation. Autophagy (literally self-eating) is a major intracellular catabolic pathway that has recently emerged as a critical process in multiple patho/physiological functions including development, aging, immunity, cancer and pathogen infection (Mizushima et al., 2008). This is the only cellular pathway responsible for the degradation

of intracellular membranes, including ER, mitochondria, and peroxisomes and, as such, it represents an important cellular process for the regulation of size and number of these organelles. Moreover, recent work demonstrates that autophagy is also involved in the degradation of large protein complexes and protein aggre-

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gates. Autophagy is considered a nonselective process serving for ‘‘bulk’’ protein and organelles degradation, but a growing body of evidence is providing clear-cut examples of molecular mechanisms for cargo specificity. For example, peroxisome recruitment into autophagosomes (pexophagy) is dependent on PEX3 and

Developmental Cell

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Figure 1. Schematic Model for the Regulation of Lipophagy (Lipid Droplets Autophagy) Under normal growth conditions, LDs are degraded at a basal level via the autophagic pathway (top). Elevation in free fatty acids stimulates LD growth and induces LD degradation by lipophagy (middle). Under stress conditions, such as starvation, the selective degradation of LDs by lipophagy increases, thus providing cells with needed nutrients (bottom). The role of the cytosolic lipases and a possible crosstalk with the autophagic process, both in basal and stimulated lipolysis, has not been determined.

PEX14, two peroxisomal proteins that are essential for targeting peroxisomes to autophagy (Farre et al., 2008). Similarly, the lysosomal degradation of mitochondria (mitophagy) requires Uth1/Aup1 and parkin, proteins needed for the selective delivery of mitochondria to autophagosomes, in yeast and mammals, respectively (for review see Tolkovsky, 2009). In ER-phagy, the expansion of the ER triggered by the unfolded protein response (UPR) is remodeled to its homeostatic volume by selective autophagy (Bernales et al., 2006). Ribophagy, which describes the selective degradation of the 60S ribosomal subunit by the autophagic pathway, is regulated by the ubiquitin protease Ubp3 and its activator Bre5 (Kraft et al., 2008). Finally, p62 and NBR1, two scaffold proteins, act as recruiters of ubiquitinlabeled protein aggregates to autophagosomes for specific degradation (Kirkin et al., 2009). The involvement of autophagy in yet another selective organelle degradation is reported in a recent issue of Nature by Singh and colleagues (2009), who demonstrated that clearance of lipid droplets (LDs) is mediated by lipid specific autophagy (lipophagy). LDs are neutral lipid storage organelles that are found in all organisms from bacteria to human (Fujimoto et al., 2008).

LDs are composed of a hydrophobic core of triglycerides (TGs) and sterol esters enwrapped by a polar lipids monolayer associated with various proteins. These unique organelles have an essential role in energy storage, as their accumulation in adipocytes comprises the largest energy reservoir for the whole body. Breakdown of LDs supplies free (nonesterified) fatty acids, which undergo b-oxidation in the mitochondria to support ATP production. In addition to their metabolic role, LDs are also involved in cellular lipid homeostasis, temporal protein storage, and protein degradation and serve as a source for signaling lipids (Thiele and Spandl, 2008). LDs are dynamic organelles, which are formed, grow, or degrade in response to various signals and physiological conditions. These cytosolic organelles originate in the ER and in adipocytes can reach a diameter of 20 mm, an enlargement driven by an unknown mechanism. Degradation of LD-sequestered TGs (lipolysis) is thought to take place mainly by cytosolic lipases under the direct control of several LD proteins, such as the PAT protein family, regulated by extracellular signaling factors like TNF and insulin (Thiele and Spandl, 2008). However, the question remains whether the regulation of size

and number of these extremely large organelles can be attributed solely to the cytosolic lipases. Czaja’s and Cuervo’s groups now demonstrate, for the first time, the involvement of autophagy in LD lipolysis in hepatocytes (Singh et al., 2009). The authors found that blocking the autophagic pathway, either by chemical or genetic approaches, leads to the accumulation of LDs. Using electron microscopy the authors showed that LDs are found within double-membrane vesicles and that LC3, an autophagosomal marker, labeled membrane structures surrounding the LDs. The presence of LC3 was taken as evidence for the formation of autophagic membranes around LDs, although a recent report by Shibata and colleagues (2009) proposed that LC3 localizes to the surface membrane of LDs and is essential for their biogenesis. According to Singh and coworkers (2009), autophagy-mediated LD clearance is augmented in response to external stimuli for LD accumulation, such as methionineand choline-deficient medium or oleate addition, suggesting selectivity toward LDs under exogenous lipid load. Conclusive evidence for the involvement of autophagy in LD hydrolysis emerges from Singh’s and colleagues in vivo experiments. Using wild-type and Atg7

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Previews knockout mice, which have defects in autophagy, the authors confirmed that the LD lipolysis in hepatocytes is autophagy dependent. Moreover, by EM analysis of wild-type mice, the authors describe three kinds of autophagosomes distinguished by their content: some contain only LDs, some contain both LDs and other cytoplasmic constituents, while others do not contain detectable LDs. Long starvation periods lead to an increase in LD-containing autophagosomes (lipophagosomes), highlighting the physiological significance of this process for survival. Furthermore, using high fat diet-fed mice, the authors suggest that autophagy is inhibited following excessive lipid consumption— a process that may have major therapeutic implications. However, further experiments are needed to elucidate this process. In summary, lipophagy may act on the one hand as a homeostatic regulator that controls the size and number of LDs under basal conditions and on the other hand as a stress-induced survival mechanism that provides the cell with energy source (Figure 1). These exciting findings suggest that a novel type of autophagy (lipophagy) specifically regulates lipid homeostasis. However, the molecular mechanism by which the autophagic

machinery recognizes LDs is not clear. Several LD resident proteins regulate TGs degradation by cytosolic lipases and thus may play a role in the selective recruitment of the autophagic machinery. It remains unclear which lysosomal lipase(s) is responsible for TG breakdown. The identification of lipid-specific autophagy by Singh and colleagues (2009) raises new questions concerning the relationship between lipophagy and canonical autophagy as well as between lipophagy and cytosolic lipolysis. Thus, exploring whether cytosolic lipolysis also plays a role in LD clearance under starvation or in response to lipid load will greatly contribute to the understanding of this field of study. Similarly, it is also important to test whether the autophagic process contributes to what has been known as a cytosolic lipolysis, induced by stimuli such as chronic exposure of adipocytes to TNF and insulin. The regulation of the LD cycle of accumulation and degradation by lipophagy may also have great physiological consequences, since an impaired cycle could lead to disorders associated with obesity and type-2 diabetes (like fatty liver diseases). Therefore, deciphering the molecular mechanism governing this process may provide new therapeutic tools.

REFERENCES Bernales, S., McDonald, K.L., and Walter, P. (2006). PLoS Biol. 4, e423. 10.1371/journal.pbio. 0040423. Farre, J.C., Manjithaya, R., Mathewson, R.D., and Subramani, S. (2008). Dev. Cell 14, 365–376. Fujimoto, T., Ohsaki, Y., Cheng, J., Suzuki, M., and Shinohara, Y. (2008). Histochem. Cell Biol. 130, 263–279. Kirkin, V., Lamark, T., Sou, Y.S., Bjorkoy, G., Nunn, J.L., Bruun, J.A., Shvets, E., McEwan, D.G., Clausen, T.H., Wild, P., et al. (2009). Mol. Cell 33, 505–516. Kraft, C., Deplazes, A., Sohrmann, M., and Peter, M. (2008). Nat. Cell Biol. 10, 602–610. Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. (2008). Nature 451, 1069–1075. Shibata, M., Yoshimura, K., Furuya, N., Koike, M., Ueno, T., Komatsu, M., Arai, H., Tanaka, K., Kominami, E., and Uchiyama, Y. (2009). Biochem. Biophys. Res. Commun. 382, 419–423. Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K., Cuervo, A.M., and Czaja, M.J. (2009). Nature, in press. Published online April 1, 2009. 10.1038/nature07976. Thiele, C., and Spandl, J. (2008). Curr. Opin. Cell Biol. 20, 378–385. Tolkovsky, A.M. (2009). Biochim. Biophys. Acta, in press. Published online March 13, 2009. 10.1016/j. bbamcr.2009.03.002.

Spreading the Silence Janet F. Partridge1,* 1Department of Biochemistry, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.05.002

RITS (RNA-induced initiation of transcriptional gene silencing complex) plays diverse roles in heterochromatin regulation: silencing transcription by recruitment of chromatin modifiers and destroying transcripts by RNAi. In a recent issue of Molecular Cell, Li et al. now show that the polymerization of Tas3, a component of RITS, contributes to the spreading of silencing mediated by RITS. One of the oldest and most interesting questions in the field of gene expression is how genes can be turned on or off dependent on their location within the genome. This effect, coined position effect variegation (PEV), relies on the ‘‘spreading’’ of states of activity or inactivity and can in some cases regulate gene expression

over tens of kilobases of DNA. A number of chromatin binding complexes have been implicated in such spreading and several distinct models of spreading have been proposed (Talbert and Henikoff, 2006). In some cases, spreading is linked to the active progression of polymerases through a locus, while in others spreading

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is more passive, ‘‘oozing’’ independent of polymerase. In such oozing models, binding of a chromatin regulatory complex is followed by reiterative cycles of multimerization and recruitment of enzymatic activity, leading to modification of adjacent nucleosomes, further binding of the complex and spreading along chromatin.