Transcriptional and post-transcriptional regulation of autophagy in the yeast Saccharomyces cerevisiae

Autophagy is a highly conserved catabolic pathway that is vital for development, cell survival, and the degradation of dysfunctional organelles and potentially toxic aggregates. Dysregulation of autophagy is associated with cancer, neurodegeneration, and lysosomal storage diseases. Accordingly, autophagy is precisely regulated at multiple levels (transcriptional, post-transcriptional, translational, and post-translational) to prevent aberrant activity. Various model organisms are used to study autophagy, but the baker's yeast Saccharomyces cerevisiae continues to be advantageous for genetic and biochemical analysis of non-selective and selective autophagy. In this Minireview, we focus on the cellular mechanisms that regulate autophagy transcriptionally and post-transcriptionally in S. cerevisiae.


Overview
In response to external environmental and internal homeostatic cues, cells must efficiently and successfully adapt to ensure survival during stress conditions. Macroautophagy/autophagy is a highly conserved (from yeast to human) catabolic mechanism of "self-eating" that is vital for homeostasis, development, and the clearance of damaged or superfluous organelles and protein aggregates, substrates that cannot be degraded by the proteasome, the other major degradative pathway in eukaryotic cells. Autophagy occurs in all eukaryotic cells (1), underlying its importance. The classical morphological feature of autophagy is the formation of the double-membrane structure termed the autophagosome. In most cells, basal autophagy generally occurs at a low level but is markedly induced in response to nutrient deprivation, pathogen infection, and other forms of stress. Autophagic flux results in the fusion of the autophagosome with the vacuole (in yeast or plants) or a lysosome (in mammalian cells). In yeast, vacuolar hydrolases degrade the autophagic cargo. This degradation is followed by efflux of the breakdown products for reuse, helping to safeguard cell survival particularly during starvation or low-energy conditions.
When induced by nutrient deprivation or pharmacological means, non-selective autophagy targets bulk cytoplasm for uptake into the phagophore, the autophagosome precursor. During selective types of autophagy, the phagophore sequesters specific cargo (such as organelles or invasive pathogens) through receptor-mediated interactions between selective autophagy receptors and Atg8 (which is located on both sides of the phagophore), and unique cargo-localized ligands, thereby generally excluding bulk cytoplasm. In addition to receptor targeting, specific cargos are further selected by proximity to the expanding phagophore membrane (2). In yeast, under nutrientrich conditions, a biosynthetic form of selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) 2 pathway, delivers resident hydrolases to the vacuole (3)(4)(5)(6). Multiple forms of selective autophagy in yeast have been characterized, including mitophagy (7,8), pexophagy (9), reticulophagy (10,11), ribophagy (12), granulophagy (13), aggrephagy (14), nucleophagy (11,15), lipophagy (16, 17), and piecemeal microautophagy of the nucleus/micronucleophagy (18,19). Although not considered to be a selective type of autophagy, the degradation of bulk RNA has also been described (20). At least in yeast, the receptors utilized to target cargos are unique to the form of selective autophagy that the cell is undergoing (5,7,8,21). For further discussion on the topic of selective autophagy, see Refs. 22,23. Autophagy is a highly complex and rigorously coordinated process; at present, 41 unique autophagy-related (ATG) genes have been identified in fungi, and many have homologs in higher eukaryotes. Autophagy dysregulation is associated with multiple human pathologies such as cancer, neurodegeneration, microbial infection and lysosomal storage diseases. Thus, autophagy must be strictly modulated to maintain an appropriate level, as too much or too little can be deleterious to the cell. Accordingly, eukaryotic cells have evolved mechanisms to tightly control and coordinate autophagy at multiple levels (transcriptional, post-transcriptional, translational, and posttranslational; see Fig. 1). In this Minireview, we focus on autophagy regulation, particularly at the transcriptional and post-transcriptional levels, in the yeast Saccharomyces cerevisiae. In addition to its high degree of conservation with other systems, S. cerevisiae continues to be particularly advantageous for genetic and biochemical analysis of non-selective and selective forms of autophagy. This is the second article in the Thematic Minireview series "Autophagy." The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1

THEMATIC MINIREVIEW
Autophagy is a dynamic process that requires constant finetuning at multiple levels to maintain the appropriate timing of induction and magnitude within the cell. Cells control the level of the autophagic response largely by regulating the size and frequency (i.e. number) of autophagosomes (24,25). Here, we will briefly describe the main phases of the autophagic process and the machinery involved before further reviewing recently published work on its transcriptional and post-transcriptional regulation.

Autophagy in S. cerevisiae
There are five main stages of autophagy, including: 1) induction and nucleation of the phagophore membrane; 2) expansion of the phagophore; 3) closure and maturation to form the autophagosome; 4) autophagosome-vacuole fusion; and 5) degradation/efflux of the breakdown products (see Fig. 2). Briefly, in yeast, autophagy is typically stimulated through nutrient deprivation (most commonly with nitrogen starvation) or with the use of a pharmacological agent such as rapamycin. Rapamycin treatment inhibits target of rapamycin (TOR), a serine/threonine kinase and major negative regulator of autophagy induction in yeast (26). In addition to TOR, upstream nutrient sensors such as protein kinase A and Snf1 (the homolog of mammalian AMP kinase) integrate signals for autophagy regulation (27). In yeast, the phagophore assembly site (PAS) is the intracellular location of autophagosome formation. At the PAS, Atg proteins assemble in a hierarchical order. The first complex recruited to the PAS includes Atg1, Atg13, and the Atg17-Atg31-Atg29 ternary subcomplex (28,29). Next, Atg9 (along with Atg2 and Atg18) localizes to the PAS (30). The phagophore, a dynamic cup-shaped membrane structure, transiently envelops bulk cytoplasm or specific cargo. This sequestration event is followed by expansion and closure of the phagophore to form the mature autophagosome and requires two conserved ubiquitin-like (Ubl) conjugation systems, which involve Atg12 and Atg8. The Atg12 Ubl system includes Atg5, Atg7, Atg10, Atg12, and Atg16 and leads to the formation of the heterotrimeric complex Atg12-Atg5-Atg16, which may function as an E3-like enzyme for the Atg8 conjugation system, although this complex is not absolutely required for conjugation to occur (31)(32)(33)(34). The conjugation of Atg12 to Atg5 at the phagophore occurs sequentially, requiring Atg7, an E1-like enzyme that activates Atg12, and Atg10, an E2-like enzyme (35,36). The Atg8 Ubl system is necessary for membrane expansion and closure of the phagophore, and this second system includes Atg3, Atg4, Atg7, and Atg8 (30). Non-lipidated Atg8 is converted to its lipidated phosphatidylethanolamine-conjugated species following Atg4-mediated proteolytic processing of its C terminus, Atg7-dependent activation, and Atg3-facilitated conjugation at a conserved C-terminal glycine (37,38).
The next major step in autophagy involves fusion between the outer membrane of the autophagosome and the vacuole. The resulting vesicle formed by this event is termed the autophagic body and consists of the remaining inner autophagosome membrane found within the vacuole lumen. The final events in autophagic flux include degradation of the cargo, followed by efflux of the resulting macromolecules. In yeast, the vacuole has enzymes for degrading the major macromolecules; however, an efflux mechanism has only been identified for amino acids (39), and catabolized RNA products appear to be secreted from the cell (20). For a more comprehensive review on the main stages of autophagy and the role of ubiquitin, please see Refs. 28, 40, 41.

Background
As mentioned above, cells must successfully fine-tune and integrate signals at multiple regulatory levels to maintain appropriate control over autophagy. Here, we focus on transcriptional and post-transcriptional regulation of autophagy in S. cerevisiae. For further discussion on autophagy regulation, particularly at the epigenetic and post-translational levels, please see these recent reviews in Refs. 40, 42-45.

Major transcriptional regulators in yeast
Ume6 -Ume6 is a DNA-binding protein that has dual functions both as a transcriptional activator and as a repressor depending on the growth conditions (46,47). Ume6 is dependent on the corepressor Sin3 and histone deacetylase Rpd3, and all are components of the multisubunit Rpd3 large (Rpd3L) complex in yeast (48 -50). Rpd3L is one of the 2 Rpd3 histone deacetylase complexes that regulate gene expression (50 -52). A Ume6 consensus-binding site (URS1 region) is found within the ATG8 promoter (53); Ume6 directly binds (and thereby represses) the ATG8 promoter under nutrient-rich conditions (53). Ume6 undergoes phosphorylation by the kinase Rim15 THEMATIC MINIREVIEW: Regulation of autophagy in yeast (further detailed below) during nitrogen starvation, which leads to the derepression of ATG8, thus promoting ATG8 transcription (53). Deletion of UME6, SIN3, and/or RPD3 significantly up-regulates ATG8 mRNA (and consequently protein) under nutrient-rich conditions, and autophagy is more rapidly induced in ume6⌬ cells during nitrogen starvation (53). Importantly, the amount of Atg8 directly correlates with the size of autophagosomes during starvation conditions (24). These findings support a mechanism whereby the cell is primed for a rapid autophagic response once it encounters starvation conditions. Although Ume6 is not conserved in more complex eukaryotes, the mammalian SIN3 proteins appear to play a similar role in regulating the expression of the Atg8 homolog MAP1LC3B (53).
Pho23-Pho23 is another member of the Rpd3L complex (50, 54) and a negative regulator of autophagy activity in yeast (25). Deletion of PHO23 results in the up-regulation of multiple ATG transcripts, including ATG1, -7-9, -12, -14, and -29 and an increased frequency of autophagosome formation (i.e. number) (25). Pho23 represses ATG9 under nutrient-replete conditions, and levels of Atg9 directly affect the frequency of autophagosome formation (25). Atg9 is the only integral membrane protein component of the core autophagy machinery-those proteins that are essential for autophagosome formation (55). Atg9 cycles between the PAS and peripheral sites close to the mitochondria during autophagy; these sites are also known as tubulovesicular clusters and are thought to correspond to donor membranes (56 -58). Phosphorylation of Atg9 at serine 122 (Ser-122) regulates anterograde movement between the peripheral sites and the PAS, thereby controlling the rate of autophagosome formation (59). These data support a model in which Atg9 functions to provide membrane or to direct membrane delivery for phagophore expansion; thus, increased levels of Atg9 (through Pho23 derepression) allow for a greater number of autophagosomes to form, which would have a direct effect on the magnitude of autophagy activity.
Rph1-Rph1 is a Jumonji C catalytic domain-containing histone demethylase (60). However, the role of Rph1 in autophagy is independent of its demethylase activity (61). Deletion of RPH1 enhances autophagy, and overexpression of Rph1 strongly inhibits autophagy and autophagosome formation (61). Rph1 functions as a negative transcriptional regulator of autophagy by repressing the expression of a subset of ATG genes under nutrient-replete conditions, particularly ATG7, but also including ATG1, -8, -9, -14, -29, and -32 (61). Furthermore, Rph1 directly regulates ATG7 by binding to its promoter; this binding does not occur when the DNA-binding domains of Rph1 are eliminated (61). As described above, Atg7 plays an essential role in the conjugation of phosphatidylethanolamine to Atg8, which is critical for autophagosome formation. Levels of Atg7 have an impact on the magnitude of the autophagic response (61, 62). When cells are starved for nitrogen, Rim15 phosphorylates Rph1, inhibiting its repression of ATG transcripts (61). KDM4A, a mammalian homolog of Rph1, has a conserved role in autophagy induction (61). Additionally, Rph1 may mediate transcriptional control over other as yet unidentified ATG genes (63).
Rim15-The Rim15 protein kinase integrates signals from the two major nutrient sensing pathways in yeast, TOR and protein kinase A, to positively regulate autophagy (64 -65).
Although not a direct transcriptional regulator, Rim15 phosphorylates the DNA-binding proteins Ume6 (53) and Rph1 (61) to influence ATG gene transcription (66); Rim15-dependent phosphorylation inhibits these transcription factors, leading to the derepression of ATG genes (61). Future studies will determine whether Rim15 mediates the phosphorylation of additional autophagy regulatory factors, particularly those involved in other aspects of transcriptional or post-transcriptional autophagy modulation.
Gcn4 -Gcn4 is a basic leucine zipper (bZIP) transcriptional activator that primarily binds to the 5Ј-TGACTC-3Ј consensus site in the promoter regions of target genes (67). Gcn4 was initially identified to function in the amino acid starvation response and has been described as a principal regulator of autophagy gene expression (68). During growing conditions, Gcn4 positively mediates delivery of precursor aminopeptidase I in the Cvt pathway (69). When cells undergo starvation, Gcn4 regulates non-selective autophagy activity through ATG gene transcription (69). Gcn4 activates ATG1, ATG13, and ATG14 gene expression during amino acid deprivation (68) and ATG1 during nitrogen starvation (69). Gcn4 controls ATG41 mRNA expression during nitrogen starvation-induced autophagy through transcriptional activation (70). Recently, Atg41 was identified to function in Atg9 cycling and the delivery of donor membrane to expand the phagophore at the PAS (70). The translation of GCN4 is stimulated by the phosphorylation of Sui2/eIF2␣ by the protein kinase Gcn2 (71). Gcn2 itself also positively regulates autophagy, presumably by its downstream THEMATIC MINIREVIEW: Regulation of autophagy in yeast effects on Gcn4-mediated ATG transcription (72,73) and possibly through the inhibition of TOR (74).
Gat1-Gat1 is another GATA-type transcription factor that has a GATA1-type zinc finger DNA-binding motif and binds to 5Ј-GATAAG-3Ј upstream activation sequence regions in the promoters of nitrogen-sensitive genes, similar to Gln3 (79). Gat1 functions as a positive factor for autophagy induction; deletion of GAT1 results in decreased autophagy activity (69). Also similar to Gln3, deletion of GAT1 significantly down-regulates ATG7-9, -29, and -32 mRNAs; however, no additive effect is observed on ATG transcript levels when both GAT1 and GLN3 are deleted in the same strain (69).
Other transcriptional regulators-Yap1 is a bZIP transcription factor with a preference for binding at promoter sites containing 5Ј-TTACTAA-3Ј sequences (80). It has been recently reported that Yap1 positively regulates transcription of the lipase gene ATG15 during starvation-induced autophagy (81). Atg15 preferentially hydrolyzes phosphatidylserine and facilitates autophagic body lysis within the vacuole (82,83). Sfl1 functions as a transcriptional repressor and activator (84,85) and has also been recently identified to function as a positive regulator of the Cvt pathway, autophagy, and ATG8 expression (69). Fyv5 negatively regulates the expression of ATG1, -8, -9, and -14, but this effect may not be direct; in contrast, the Cvt pathway appears to be positively affected by this factor (69). Spt10, a histone H3 acetylase, represses ATG8 and ATG9 at both the RNA and protein levels (69). Although key transcriptional regulators have been identified to affect core ATG gene transcripts, the potential exists for novel factors to be discovered, particularly those that may target selective autophagy pathways.

Background
Although novel transcriptional mediators of autophagy have recently been identified (25,53,61,69), post-transcriptional regulation of autophagy in yeast is largely uncharacterized. In mammals, non-coding RNAs such as microRNAs (miRNAs) and RNA-binding proteins can modulate autophagy at the post-transcriptional level (42, 45, 86 -89). However, the RNA interference system (which is required for miRNA processing (90,91)) is not present in S. cerevisiae (92). Alternatively, another mechanism whereby cells exert post-transcriptional control over gene expression is through RNA decay pathways (which can degrade transcripts in either the 5Ј to 3Ј or the 3Ј to 5Ј direction). During canonical 5Ј to 3Ј degradation, transcripts undergo a reversible process known as deadenylation (which removes the 3Ј poly(A) tail), followed by decapping. The decapping enzyme Dcp2 removes the 5Ј-methylguanosine cap of the mRNA, resulting in an exposed 5Ј-monophosphate. Finally, the decapped cytoplasmic mRNAs undergo 5Ј-to 3Ј-mediated degradation by the cytoplasmic exoribonuclease Xrn1.

Dcp2 and RCK family RNA helicases Dhh1/Vad1/DDX6
A role has recently been described for Dcp2 and RCK family RNA-binding proteins as post-transcriptional regulators of ATG mRNAs, autophagy, and autophagy-dependent innate immune responses in yeast and mammalian cells (93). RCK family members-Dhh1 in S. cerevisiae, Vad1 in Cryptococcus neoformans, and DDX6/p54 in mammals-are RNA helicases acting in part as decapping accessory factors that interact with target mRNAs through recruitment to the decapping complex by binding the 5Ј-and/or 3Ј-untranslated region of selected transcripts (40,94). Dhh1 also physically interacts with the decapping enzyme Dcp2 (95).
In the report by Hu et al. (96), deletion of DHH1 or a temperature-sensitive (ts) mutation of DCP2 caused a significant up-regulation of ATG transcripts. In particular, of the ATG transcripts that were examined in this study, dhh1⌬ cells demonstrated significantly increased levels of ATG3, -7, -8, -19, -20, -22, and -24 mRNA under nutrient-replete conditions (96). In the dcp2-7⌬ ts strain, transcript levels for ATG1 through ATG9, ATG11, ATG13 through ATG24, and ATG29, -31, -32, and -34 were significantly up-regulated in rich conditions (96). Both the dhh1⌬ and dcp2-7⌬ ts strains demonstrated higher levels of autophagy activity through multiple assays when starved for nitrogen (96). This mechanism is highly conserved across the two fungal species examined (S. cerevisiae and the pathogen C. neoformans) and up through mammalian cells (96). In C. neoformans, the Dhh1 homolog Vad1 mediates decapping of ATG mRNAs, especially ATG8 (96). Furthermore, Hu et al. (96) demonstrated that Dcp2 is phosphorylated by TOR under nutrient-rich conditions in C. neoformans, which drives its association with (at least) ATG8 mRNA, stimulating recruitment of the transcript to the decapping machinery, followed by subsequent decapping. Thus, TOR acts as a negative regulator of the translation of ATG genes under conditions where it promotes the translation of the vast majority of cellular transcripts. In mammalian cells, mechanistic TOR phosphorylates DDX6, the homolog of Dhh1/Vad1, resulting in decapping of the MAP1LC3B transcript under nutrient-rich conditions to repress autophagy (96). Transcripts are then presumably degraded to maintain autophagy at a basal level. In contrast, starvation conditions inactivate TORC1, arresting degradation of the targeted transcripts and promoting sustained autophagy (96).

Xrn1
The data on Dhh1/Vad1 and Dcp2 imply a role for 5Ј to 3Ј mRNA degradation in autophagy regulation. Recent work shows that the RNase Xrn1/XRN1, which functions downstream of the decapping complex in the canonical 5Ј to 3Ј RNA THEMATIC MINIREVIEW: Regulation of autophagy in yeast decay pathway, is also a negative post-transcriptional regulator of autophagy in both yeast and mammalian cells (97). Chromosomal deletion of XRN1 induces a more rapid and robust autophagy response as determined through multiple assays in yeast (97). We also found that the frequency of autophagosome formation increases in starved xrn1⌬ cells compared with wildtype cells based on transmission electron microscopy (97). Furthermore, when xrn1⌬ cells are assessed by quantitative PCR under nutrient-rich conditions, select ATG transcripts are found to be up-regulated, including ATG1, -4, -5, -7, -8, -12, -14, -16, -29, and -31. Regulation of (at least) ATG8, ATG12, and ATG29 is dependent upon the RNase activity of Xrn1 (97).
In mammalian cells, there is enhanced autophagy activity in a starvation-independent manner when XRN1 is depleted by small interfering RNA (97). The impact on autophagy is blocked in BECN1 or ATG5 CRISPR knockout cells, supporting the role of the canonical autophagy pathway (97); BECN1 is a component of the phosphatidylinositol 3-kinase complex required for autophagy induction (98). In addition, reduction of XRN1 levels is associated with an up-regulation of poliovirus infection in an autophagy-dependent manner (97), underlying the role of Xrn1/XRN1 as a conserved autophagy regulator. Poliovirus, similar to other picornaviruses, utilizes host membranes that are proposed to be derived from autophagosomes to support viral genome replication (97,99,100).

Summary
The studies by Hu et al. (96) and Delorme-Axford et al. (97) provide a paradigm for post-transcriptional autophagy regulation, but they also represent the limit of what is known about this type of regulation of autophagy in yeast. Although these findings are exciting and novel, it is still not clear why there appears to be differential targeting of transcripts in the strains examined (dcp2-7⌬ ts, dhh1⌬, and xrn1⌬) even though they encode components of a related pathway of RNA degradation (96,97). Thus, despite these insights, much work remains to further understand how autophagy is controlled post-transcriptionally. For example, how are transcripts selected for degradation under nutrient-replete conditions? Although Dhh1 and Xrn1 are likely to drive some degree of specificity, many more ATG transcripts are affected in the dcp2-7⌬ ts cells compared with the dhh1⌬ and xrn1⌬ strains (96,97). Based on these data, Dhh1 and Xrn1 are likely not the only factors mediating selective transcript targeting. Furthermore, although S. cerevisiae does not post-transcriptionally regulate gene expression through miRNA, additional non-coding RNA mechanisms such as long non-coding RNA could potentially play a role in autophagy regulation. Future studies should be aimed toward uncovering additional components moderating ATG gene expression through RNA decay.

Conclusions
Here, we present a concise review of what is currently known regarding the transcriptional and post-transcriptional regulation of autophagy in the yeast S. cerevisiae (see Fig. 3 for a summary). Recent advances in the field have provided additional clues as to the myriad of regulatory mechanisms that cells maintain to tightly control and coordinate autophagy. How-ever, many questions remain, particularly concerning how autophagy is regulated post-transcriptionally, not only in yeast but in other eukaryotic systems as well. Additional novel posttranscriptional regulatory factors have yet to be identified and characterized. Further investigation into the mechanisms by which cells control the major stages of autophagy-especially induction and magnitude-at multiple regulatory levels is critical to enhance our understanding of how this essential process is maintained in the cell to promote normal physiological processes and how its dysregulation contributes to disease pathogenesis.
THEMATIC MINIREVIEW: Regulation of autophagy in yeast