Autophagosomes are organelles capable of sequestering and degrading diverse cytoplasmic cargo for nutritional and quality control purposes. Targeted are also lipid droplets (LDs), the cytoplasmic stores of neutral lipids. In this issue of The EMBO Journal, Shpilka et al (2015) show that the relationship between LDs and autophagosomes is far more intricate and that LDs regulate autophagosome biogenesis.
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Autophagosomes (Mizushima et al, 2011) and lipid droplets (LDs) (Zechner et al, 2012) are intriguing cytoplasmic organelles that, based on their fundamental makeup differences, could not appear to be further apart. Autophagosomes are double‐membrane (four leaflets) organelles with a lumen filled with cytosol or more complex cytoplasmic components. In contrast, LDs are delimited by a hemi‐membrane composed of just one leaflet/phospholipid monolayer and are filled with fat, comprised of neutral lipids, triglycerides/triacylglycerols (TGs), and cholesterol esters. Nonetheless, several recent studies (Singh et al, 2009; Velikkakath et al, 2012; Dupont et al, 2014; Rambold et al, 2015; Shpilka et al, 2015) have connected autophagosomes and LDs in a variety of ways (Fig 1).
One of the more widely accepted relationships is that autophagosomes can engulf LDs (Fig 1, pathway 1) in a process termed “lipophagy” (Singh et al, 2009) with the outcome being a lysosomal lipolysis and release of fatty acids for metabolic needs. The majority of studies have supported, albeit others have contested (Zechner et al, 2012) this view in the overall metabolic mobilization of fat, lipolysis, and fatty acid β‐oxidation. Recent work (Dupont et al, 2014; Rambold et al, 2015) has indicated that autophagosomes and LDs may have more surreptitious relationships than the simplistic view of “autophagosomes eat LDs”. One of these studies (Rambold et al, 2015) shows that during starvation, which is a classical inducer of autophagosome formation, the lipolysis is helped by autophagy but occurs not through “lipophagy”. Instead, lipolysis occurs through a classical pathway of mobilizing TGs through the adipose triacylglycerol lipase ATGL, which acts enzymatically on the surface of LDs (Zechner et al, 2012). Nevertheless, autophagy helps sustain this process by turning over other cytoplasmic components and, in the model proposed (Rambold et al, 2015), somehow replenishes TGs in LDs (Fig 1, pathway 2). The study by Shpilka et al (2015) goes much further than any of the earlier reports and flips the order in the relationship between LDs and autophagy: instead of being a substrate for lipophagy, LDs act as contributors to autophagosome biogenesis (Fig 1, pathway 3).
In their elegant study employing the power of yeast genetics, Shpilka et al (2015) demonstrate that enzymes essential for formation of LDs and responsible for TG (Dga1 and Lro1) and steryl‐ester (Are1 and Are2) synthesis are also required for functional autophagy. This observation alone would be highly intriguing and raises many interesting questions. However, Shpilka et al (2015) go a step further to determine that the newly identified yeast TG lipase Ayr1 and the lipase Ldh1 (but not the well‐established TG lipases Tgl3, Tgl4, Tgl5) are essential for autophagy. One plausible interpretation of these findings is that TGs stored in LDs need to be mobilized in a specific fashion to enable autophagosome biogenesis. Since exogenously added fatty acids could not rescue autophagy in an LD‐deficient strain of yeast, the requirement for LDs is not simply as a source of fatty acids and energy to support autophagosome biogenesis. Furthermore, Shpilka et al (2015) have shown that in addition to TG mobilization, steryl‐ester mobilization through enzymes Tgl1, Yeh1, and Yeh2 is just as important. Finally, Ldh1, which has both TG and steryl‐ester lipase/hydrolase activities, synergizes with the requirement for the TG lipase Ayr1, once again pointing to the necessity to mobilize all neutral lipid components of LDs to support autophagy. Of note, in contrast to the starvation‐induced bulk autophagy, which requires large autophagosomes, the tighter, smaller autophagic organelles of the Cvt pathway did not show these requirements, indicating that it is an increased demand for lipids in the biogenesis of spacious autophagosomes that may be particularly sensitive to the ability to mobilize neutral lipids from LDs.
Finally, Shpilka et al (2015) show that ER–lipid droplet contact sites were important in these processes. Ice2 and Ldb16, which reside in the contact sites, couple TG utilization with lipid synthesis in the ER. They also control phospholipid synthesis and lipid droplet size determined by the phospholipid/neutral lipid ratios (Wang et al, 2014). Shpilka et al (2015) found that absence of either Ice2 or Ldb16 diminished starvation‐induced autophagy. This resonates with the significance of the ER in autophagosome biogenesis, which is believed to be a major source for autophagosomal membranes. It furthermore underscores the role of LDs as an important contributor to autophagosome formation, since LDs emerge from the ER where TG synthesis occurs.
The study by Shpilka et al (2015) is not only elegant but also a very definitive demonstration of the precursor–product relationship between LDs and autophagosomes. In keeping with the result by Shpilka et al (2015) in yeast, Dupont et al (Dupont et al, 2014) have reported similar relationships in mammalian cells. In mammalian cells, the reserves of neutral lipids in LDs are needed for optimal autophagy of a variety of cargo (autophagic receptors, long‐lived proteins, mitochondria, and invading microbes such as M. tuberculosis). Contacts between LDs and autophagosomes have been observed (Yla‐Anttila et al, 2009; Dupont et al, 2014), and the two organelles engage in kiss‐and‐run events (Dupont et al, 2014) that may allow direct exchange of the material. What might be the material that is exchanged in the transient kiss‐and‐run events (Dupont et al, 2014) or by the proposed reverse flow of lipids to the ER and then to autophagosomes (Shpilka et al, 2015)? A relevant type of molecules would be phospholipids or their precursors such as diacylglycerol (DAG) liberated from TGs by the action of lipases, as inferred by Shpilka et al (2015) in yeast. Indeed, a similar screen for TG‐mobilizing enzymes in mammalian cells has identified PNPLA5, a patatin‐like phospholipase domain‐containing protein and a member of the PNPLA1‐PNPLA5 family (ATGL is PNPLA2), as co‐localizing with the autophagic membrane factor ATG16L1 on LDs and playing a role in autophagy initiation/autophagosome elongation. This includes effects on conjugation of LC3 (mammalian Atg8) to phosphatidylethanolamine, a key event in autophagy best known as the conversion from LC3‐I into its lipidated LC3‐II form. In agreement with these observations, Shpilka et al (2015) found that during starvation, yeast Atg8 accumulated in its unlipidated form in the tagΔ mutant (dga1Δ lro1Δ) defective in TG synthesis. Intriguingly, it was the lipidated Atg8 form that accumulated in the steΔ and the steΔtagΔ strains defective in steryl‐ester synthesis. Shpilka et al (2015) provide a tantalizing albeit tentative explanation for this as a possible influence of LDs on the lipidation and de‐lipidation of Atg8, which apparently undergoes a rapid regulatory cycling between lipid‐conjugated and deconjugated forms mediated by Atg4 (Nair et al, 2012).
These studies reveal a wealth of interactions between LDs and autophagosome biogenesis, with exciting possibilities for regulatory interactions as well as bulk lipid flow. According to Shpilka et al (2015), this lipid flow may go from LDs back to the ER from where they are mobilized into autophagosomal membranes, whereas others (Dupont et al, 2014) suggest that there may be an additional, direct kiss‐and‐run exchange between LDs and autophagosomes, whereby the LDs donate lipids to the outside membrane of the phagophore, possibly contributing to curvature through asymmetric loading from the phospholipid monolayer of the lipid droplet or by donating DAG as a curvature‐inducing lipid.
It is enlightening to see the latest developments in the relationship between LDs and autophagosomes. A further twist in these interconnections is that autophagy factors have been directly (i.e. independently of the process of autophagy) implicated in controlling the size of LDs (Velikkakath et al, 2012). The latest studies also underscore the role of heterotypic inter‐organellar contact sites (Yla‐Anttila et al, 2009; Dupont et al, 2014; Rambold et al, 2015; Shpilka et al, 2015), a popular topic in cell biology in general. The attractiveness of these concepts is not in the controversy but in being non‐mutually exclusive, simply revealing multiple intersections between LDs and autophagy (Singh et al, 2009; Dupont et al, 2014; Shpilka et al, 2015) along with mitochondria (Rambold et al, 2015) and the ER. Given the role of lipid metabolism and autophagy in many disease‐associated processes, understanding this particular set of relationships may be of potentially high payoff value.
- © 2015 The Author