PerspectiveCell Biology

New Targets for Acetylation in Autophagy

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Science Signaling  03 Jul 2012:
Vol. 5, Issue 231, pp. pe29
DOI: 10.1126/scisignal.2003187


Macroautophagy is an evolutionarily conserved homeostatic process that mediates the degradation of long-lived cytoplasmic components in eukaryotes, which allows cells to survive stresses such as inflammation, hypoxia, and deprivation of nutrients or growth factors. At least 30 members of the Atg (autophagy-related) protein family orchestrate this degradative process. Additional complexity resides in the signaling networks controlling the autophagic process, which include various posttranslational modifications of key components. Evidence is accumulating that protein acetylation represents an evolutionarily conserved mechanism tightly regulating macroautophagy.

Macroautophagy (referred to hereafter simply as autophagy) is a homeostatic and conserved eukaryotic process that ensures the degradation of cytoplasmic components through a lysosomal pathway (1). Autophagy, which has been described as a “self-eating” process, acts in most cells at a basal rate as a cytoplasmic quality-control mechanism, eliminating protein aggregates and damaged organelles to maintain tissue homeostasis (2). Furthermore, malfunctioning autophagy has been observed in many human diseases, including cancer, neurodegenerative and cardiovascular disorders, and infectious and inflammatory diseases (3).

The multistep autophagic process starts with the formation of a double-membraned vacuole, known as an autophagosome, which engulfs damaged proteins and organelles. The autophagosome then fuses with the endocytic compartment and ultimately with lysosomes, which results in the degradation of the sequestered material (4). The proteasome is another cellular mechanism of degradation but is limited to proteins. Only autophagy can degrade long-lived macromolecules and entire organelles and thus ensures the turnover of cellular materials and the recycling of multiple types of intracellular constituents (amino acids, fatty acids, and nucleotides) (5). During starvation, nutrient deprivation, growth factor depletion, or hypoxia, autophagy is an important stress response–induced process that maintains metabolism—and thus adenosine triphosphate concentration—and that contributes to cell survival. Autophagy involves the hierarchical assembly and coordinated actions of products of the Atg family of genes. The Atg proteins and their binding partners orchestrate all the steps from the generation of preautophagosomal structures (PASs) to the expansion of the autophagosomes, then the fusion of the autophagosomes with lysosomes (Fig. 1) (6).

Fig. 1 Acetylation of core autophagy components in the regulation of autophagy.

(A) An overview of the steps in autophagy, showing the formation of a PAS to which Atg proteins are hierarchically recruited to form the isolation membrane of phagophore and then the enclosed autophagosome. Maturation and fusion with lysosomes result in the formation of autolysosomes and ultimately release of the digested contents. (B) Reversible cytoplasmic acetylation of core autophagy components regulates the autophagosome formation in yeast and mammalian cells. In nitrogen-starved yeast, Esa1 acetylates Atg3 and promotes the conjugation of Atg8 to PE and its incorporation into expanding autophagosomes. Delayed deacetylation of Atg3 by starvation-activated Rpd3 attenuates the autophagic response (13). In mammalian cells deprived of serum, TIP60 is activated and acetylates ULK1, an initiator of formation of the PAS. This pathway functions in the absence of serum but is not needed for autophagy in cells deprived of glucose. On the basis of conservation with yeast, TIP60 may stimulate the acetylation of ATG3 to initiate serum starvation–induced autophagy (broken arrow) (14). Also shown are the hierarchical Atg complexes that contribute to the formation of the autophagosome starting with the ULK1 (Atg1) complex (orange), the ATG14 complex (blue), and the ATG5 complex (purple). EPL-1, Enhancer of Polycomb-like-1; hVPS34, human class III phosphatidylinositol 3-kinase.


This complex process must respond appropriately to different types of stimuli and is tightly regulated by various posttranslational modifications. Posttranslational modifications modulate the activity of the autophagic core component proteins and thus influence the rate at which autophagy occurs, which is known as autophagic flux. Ubiquitylation of autophagic cargo, such as proteins and damaged mitochondria, is needed for their selective sequestration by the autophagosomal membrane (7). Phosphorylation of Atg1p [in yeast (8)] (ULK1 in mammals) and dephosphorylation of Atg13 are crucial in triggering autophagy (6). In mammalian cells, Atg5 activation by ubiquitylation-like conjugation to Atg12 is required for autophagosomal membrane expansion (6). Posttranslational lipidation of Atg8p (LC3 in mammals) with phosphatidylethanolamine (PE), mediated by the E2-like enzyme Atg3, is also necessary for the expansion of the autophagosomes (9). Protein lysine acetylation is emerging as an evolutionarily conserved metabolic regulatory mechanism involved in coordinating different metabolic pathways, including autophagy, in response to extracellular conditions (10). For example, Atg5, Atg7, Atg8, and Atg12 have been reported as acetylated (7), and acetylation of Atg proteins can either promote or inhibit their function in autophagy.

In mammalian cells under nutrient-rich conditions, the acetyltransferase p300 directly interacts with Atg7 and acetylates the autophagy proteins Atg5, Atg7, Atg8, and Atg12 to inhibit autophagy (11). In contrast, during starvation, p300 dissociates from Atg7, and the deacetylase Sirt1 removes acetyl groups from the Atg7, Atg5, Atg12, and Atg8, which allows autophagy to proceed (12). Aspects of the phenotype of Sirt1 knockout mice resemble those of Atg5 knockout mice, which suggests that Sirt1-dependent deacetylation could be important for basal autophagy and neonatal survival (12). Thus, acetylation of key autophagic proteins modulates the autophagic response.

Another acetylase has been implicated in the regulation of autophagy by enhancing another posttranslational modification, protein lipidation. Esa1p in Saccharomyces cerevisiae (13) and its mammalian ortholog TIP60 (14) are histone acetylases that function as evolutionarily conserved regulators of autophagy. Yi et al. (13) identified Atg3p, which is necessary for Atg8p lipidation, as a substrate of Esa1p during starvation-induced autophagy. Lysines 19, 48, and 183 of Atg3p were targets for Esa1p-mediated acetylation. Acetylation of Lys183 enhanced the lipid-conjugating activity of Atg3p, and acetylation of Lys19 and Lys48 promoted the interaction between Atg3p and Atg8p, which is necessary for the conjugation of Atg8p to PE (15). It would be interesting to determine whether acetylation of Atg3p modifies its recognition of the substrate PE, which is critical for conjugation of PE to Atg8p (16).

Yi et al. (13) identified a temporal regulatory mechanism controlling the duration and magnitude of autophagy through reversible acetylation of Atg3p that occurred during starvation-induced autophagy. Using genetic analysis, they identified the histone deacetylase Rpd3p as attenuating the starvation-induced acetylation of Atg3p. Thus, Atg3p acetylation is transient, which contributes to the attenuation of the formation of autophagosomes during starvation. Although Esa1p and Epl1p (the catalytic and regulatory subunits of the NuA4 histone acetyltransferase complex) and Rpd3p are mainly located in the nucleus, a fraction of each is recruited to the PAS during starvation. The recruitment of Rpd3p to the PAS occurs after a delay, which allows the acetylation of Atg3p by Esa1p and the initiation of autophagy to occur before the process is attenuated by Rpd3p. Thus, Atg3 acetylation is subject to both spatial and temporal regulation during starvation. The mechanisms responsible for the recruitment of the Esa1p, Epl1p, and Rpd3p to the PAS during starvation—thus controlling the acetylation of Atg3 and the initiation, duration, and magnitude of autophagy—remain the subjects of further investigation.

Whereas Yi et al. (13) found an intersection between acetylation and lipidation in yeast, Lin et al. (14) identified crosstalk between acetylation and phosphorylation in the regulation of autophagy in mammalian cells. Lin et al. identified the acetyltransferase TIP60 as a positive regulator of autophagy in response to growth factor or serum deprivation in mammalian cells (14). Whereas Yi et al. (13) reported an interaction between Atg3p and Esa1p, Lin et al. (14) reported that TIP60 interacted with ULK1 (a mammalian ortholog of Atg1p) and failed to detect an interaction between TIP60 and ATG3 in response to serum deprivation in human colon cancer cells. Instead, Lin et al. found that, in response to growth factor deprivation, TIP60 was activated by phosphorylation at Ser86 by glycogen synthase kinase-3 (GSK3), and this phosphorylation depended on prior phosphorylation at Ser90. The dually phosphorylated TIP60 exhibited higher affinity for ULK1 than did nonphosphorylated TIP60, which resulted in an increase in the acetylation and kinase activity of ULK1 and, thus, induced autophagy in response to growth factor withdrawal. One way to resolve the discrepancy in the targets of yeast Esa1p and mammalian TIP60 would be to assess the acetylation of ATG3 and ULK1 in TIP60-silenced cells or of Atg3p and Atg1p in Esa1-deficient cells.

The study by Lin et al. (14) also provides additional evidence that GSK3 is a sensor for growth factor signaling, similar to the activity of AMPK [adenosine monophosphate (AMP)–activated protein kinase] and mTOR (mammalian target of rapamycin) as sensors for energy and nutrients, respectively (17). Thus, there is an autophagy-activating pathway composed of GSK3, TIP60, and ULK1 that integrates protein phosphorylation and acetylation to connect growth factor deprivation to autophagy. Further investigation is required to identify the kinase and the signaling pathway that regulate the phosphorylation of TIP60 on Ser90.

Lin et al. (14) reported in vivo temporal regulation of TIP60 Ser86 phosphorylation in the hearts of postnatal mice, which correlated with autophagy that occurs during the first few days after birth. Consistent with an essential role of TIP60 in autophagic induction, TIP60−/− mouse blastocysts failed to undergo implantation and died around embryonic day 3.5 at the time when autophagic activity is high during normal implantation (2, 18).

In addition to cytoplasmic acetylation reactions, autophagy can also be regulated by acetylation of nuclear proteins and thus can influence the expression of genes encoding proteins involved in autophagy. These nuclear targets of acetylation-mediated regulation include transcription factors, such as Foxo3 (19), and histones (20, 21). Resveratrol, an activator of the deacetylase Sirt1, and spermidine, an inhibitor of histone acetylases, influence the acetylation-modified proteome, induce autophagy, and increase longevity in yeast, nematodes, and flies (20, 21). Changes in the acetylation status of >100 proteins that form part of the central network of autophagic regulators or executors have been identified after treatment with resveratrol and spermidine (21).

Clearly, acetylation is emerging as an important regulatory mechanism in autophagy and various multiple metabolic pathways. Acetylation influences autophagy at multiple levels, including the modification of the autophagy core proteins, autophagic target substrates, microtubules, transcriptional factors, and histones. In addition, the acetylation of autophagy substrates can promote their lysosomal degradation (22). Acetylation joins phosphorylation, ubiquitylation, and lipidation in the complex regulatory network controlling autophagy.

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