Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy

Autophagy is a highly conserved degradative process in eukaryotic cells. This process plays an integral role in cellular physiology, and the levels of autophagy must be precisely controlled to prevent cellular dysfunction. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating the induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that Tap42 and protein phosphatase type 2A (PP2A) are involved in the regulation of autophagy in yeast. Temperature-sensitive mutant alleles of TAP42 revealed that autophagy was induced without inactivation of TORC1. Absence of the Tap42-interacting protein Tip41 abolished autophagy induction in the tap42 mutants, whereas overexpression of Tip41 activated autophagy. Furthermore, inactivation of PP2A stimulated autophagy and overexpression of a catalytic subunit of PP2A blocked rapamycin-induced autophagy. Our data support a model in which autophagy is negatively regulated by the Tap42-PP2A pathway.     

Ksp1 kinase regulates autophagy via the target of rapamycin complex 1 (TORC1) pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.     

PKA compartmentalization links cAMP signaling and autophagy

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.Subject terms: Kinases, Autophagy     

The regulation of autophagy in eukaryotic cells: do all roads pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be incompatible with rapid cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signaling pathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.     

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.     

Atg27 is a Second Transmembrane Cycling Protein

Autophagy is a degradative pathway conserved among all eukaryotic cells, and is responsible for the turnover of damaged organelles and long-lived proteins. The primary morphological feature of autophagy is the sequestration of cargo within a double-membrane cytosolic vesicle called an autophagosome. More than 25 AuTophaGy-related (ATG) genes that are essential for autophagy have been identified from the yeast Saccharomyces cerevisiae. Despite the identification and characterization of Atg proteins, it remains a mystery how the double-membrane vesicle is made, what the membrane source(s) are, and how the lipid is transported to the forming vesicle. Among Atg proteins, Atg9 was the only characterized transmembrane protein required for the formation of double-membrane vesicles. Evidence has been obtained in yeast and mammalian cells for Atg9 cycling between different peripheral compartments and the phagophore assembly site/pre-autophagosomal structure (PAS), the proposed site of organization for autophagosome formation. This cycling feature makes Atg9 a potential membrane carrier to deliver lipids that are used in the vesicle formation process.2 Recently, in our lab we characterized a second transmembrane protein, Atg27. The unique localization and cycling features of Atg27 suggest the involvement of the Golgi complex in the autophagy pathway. In this addendum, we discuss the trafficking of Atg27 in yeast and compare it with that of Atg9, and consider the possible meaning of Atg27 Golgi localization.

Addendum to:

Atg27 is Required for Autophagy-Dependent Cycling of Atg9

W.-L. Yen, J.E. Legakis, U. Nair and D.J. Klionsky

Mol Biol Cell 2006; In press     

Feedback regulation between autophagy and PKA

Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.     

The Regulation of Autophagy in Eukaryotic Cells: Do All Roads Pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be refractory to cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signalingpathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.     

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.     

Yeast chronological lifespan and proteotoxic stress: is autophagy good or bad?

Autophagy, a highly conserved proteolytic mechanism of quality control, is essential for the maintenance of metabolic and cellular homoeostasis and for an efficient cellular response to stress. Autophagy declines with aging and is believed to contribute to different aspects of the aging phenotype. The nutrient-sensing pathways PKA (protein kinase A), Sch9 and TOR (target of rapamycin), involved in the regulation of yeast lifespan, also converge on a common targeted process: autophagy. The molecular mechanisms underlying the regulation of autophagy and aging by these signalling pathways in yeast, with special attention to the TOR pathway, are discussed in the present paper. The question of whether or not autophagy could contribute to yeast cell death occurring during CLS (chronological lifespan) is discussed in the light of our findings obtained after autophagy activation promoted by proteotoxic stress. Autophagy progressively increases in cells expressing the aggregation-prone protein α-synuclein and seems to participate in the early cell death and shortening of CLS under these conditions, highlighting that autophagic activity should be maintained below physiological levels to exert its promising anti-aging effects.     

Atg27 is a second transmembrane cycling protein

Autophagy is a degradative pathway conserved among all eukaryotic cells, and is responsible for the turnover of damaged organelles and long-lived proteins. The primary morphological feature of autophagy is the sequestration of cargo within a double-membrane cytosolic vesicle called an autophagosome. More than 25 AuTophaGy-related (ATG) genes that are essential for autophagy have been identified from the yeast Saccharomyces cerevisiae. Despite the identification and characterization of Atg proteins, it remains a mystery how the double-membrane vesicle is made, what the membrane source(s) are, and how the lipid is transported to the forming vesicle. Among Atg proteins, Atg9 was the only characterized transmembrane protein required for the formation of double-membrane vesicles. Evidence has been obtained in yeast and mammalian cells for Atg9 cycling between different peripheral compartments and the phagophore assembly site/preautophagosomal structure (PAS), the proposed site of organization for autophagosome formation. This cycling feature makes Atg9 a potential membrane carrier to deliver lipids that are used in the vesicle formation process. Recently, in our lab we characterized a second transmembrane protein, Atg27. The unique localization and cycling features of Atg27 suggest the involvement of the Golgi complex in the autophagy pathway. In this addendum, we discuss the trafficking of Atg27 in yeast and compare it with that of Atg9, and consider the possible meaning of Atg27 Golgi localization.     

Mitophagy, mitochondrial dynamics and the general stress response in yeast

Autophagy is a fundamental cellular process promoting survival under various environmental stress conditions. Selective types of autophagy have gained much interest recently as they are involved in specific quality control mechanisms removing, for example, aggregated proteins or dysfunctional mitochondria. This is considered to counteract the development of a number of neurodegenerative disorders and aging. Here we review the role of mitophagy and mitochondrial dynamics in ensuring quality control of mitochondria. In particular, we provide possible explanations why mitophagy in yeast, in contrast with the situation in mammals, was found to be independent of mitochondrial fission. We further discuss recent findings linking these processes to nutrient sensing pathways and the general stress response in yeast. In particular, we propose a model for how the stress response protein Whi2 and the Ras/PKA (protein kinase A) signalling pathway are possibly linked and thereby regulate mitophagy.     

In vivo reconstitution of autophagy in Saccharomyces cerevisiae

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.     

Introducing fluorescence resonance energy transfer‐based biosensors for the analysis of cAMP‐PKA signalling in the fungal pathogen Candida glabrata

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP‐PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP‐PKA activity in this pathogen, we here present the usage of two FRET‐based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time‐resolved manner, as we exemplify by glucose‐induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP‐PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.     

The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Autophagy is a conserved, degradative process that has been implicated in a number of human diseases and is a potential target for therapeutic intervention. It is therefore important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. The Atg1 protein kinase is a key element of this control as a number of signaling pathways target this enzyme and its associated protein partners. These studies have established that Atg1 activities are controlled, at least in part, by protein phosphorylation. To further this understanding, we used a combined mass spectrometry and molecular biology approach to identify and characterize additional sites of phosphorylation in the Saccharomyces cerevisiae Atg1. Fifteen candidate sites of phosphorylation were identified, including nine that had not been noted previously. Interestingly, our data suggest that the phosphorylation at one of these sites, Ser-34, is inhibitory for both Atg1 kinase activity and autophagy. This site is located within a glycine-rich loop that is highly conserved in protein kinases. Phosphorylation at this position in several cyclin-dependent kinases has also been shown to result in diminished enzymatic activity. In addition, these studies identified Ser-390 as the site of autophosphorylation responsible for the anomalous migration exhibited by Atg1 on SDS-polyacrylamide gels. Finally, a mutational analysis suggested that a number of the sites identified here are important for full autophagy activity in vivo. In all, these studies identified a number of potential sites of regulation within Atg1 and will serve as a framework for future work with this enzyme.     

Autophagy induced by rapamycin and carbon-starvation have distinct proteome profiles in Aspergillus nidulans

It is hypothesized that autophagy, a global catabolic pathway which is highly conserved from yeast to man, plays an important role in many bioprocesses. Though autophagy is known to be induced by either nutrient starvation or treatment with the drug rapamycin, it is not clear whether the two modes of induction have the same long-term impact in the cell, particularly in the biotechnologically important filamentous fungi. Here, we compare the overall proteomes from the carbon-starved (G-) and rapamycin treated (R+) model fungus Aspergillus nidulans. From about 1,100 visualized protein spots, we conservatively selected a total of 26 proteins with significant different expression. To highlight, increased levels of glucosidases and decreased levels of N-acetylglucosamine pyrophosphorylase were observed, suggesting degradation of the fungal cell wall as an alternate carbon source for both modes of induction. Cdc37 was reduced in expression while 14-3-3 ArtA was increased, implying regulation of polar growth, while also potentially regulating autophagy negatively via PKA or Tor. Other proteins included aspartate transaminase, tryptophan synthase B (TrpB), glycylpeptide N-tetradecanoyltransferase (Nmt1), and aldehyde dehydrogenase (aldA). More interestingly, the majority of the identified proteins (16 of 26) were uniquely expressed in elevated levels in G-. A novel predicted protein from AN8223 which has no sequence homology to other organisms is also implicated to be involved in carbon-starvation. Thus, proteomic data here show that in A. nidulans, rapamycin-induced autophagy and carbon-starvation induced autophagy share some effectors for cell survival, but predominantly involve different long-term effectors.     

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A₁ (BafA₁), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA₁. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.