A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy

Autophagy is an early cellular event during acute pancreatitis, a disease defined as pancreas self-digestion. The Vacuole Membrane Protein 1 (VMP1) is a trans-membrane protein highly activated in acinar cells early during pancreatitis-induced autophagy and it remains in the autophagosomal membrane. We have shown that VMP1 expression is able to trigger autophagy in mammalian cells, even under nutrient-replete conditions. VMP1 is induced by autophagy stimuli and its expression is required for autophagosome development. VMP1 interacts with Beclin 1 through its hydrophilic C-terminal region, which we named Atg domain, as it is essential for autophagy. Remarkably, VMP1 pancreas-specific transgenic expression in mice promotes autophagosome formation. Most of the autophagy-related proteins were described in yeast or have a yeast homologue. VMP1 does not have any known homologue in yeast but its expression is required to start the autophagic process in mammalian cells. These findings support the hypothesis that mammalian cells may regulate autophagy in a different way. We propose that VMP1 is a novel autophagy related trans-membrane protein, which may lead the way in the search for alternative mechanisms of autophagosome formation.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

The TP53INP2 Protein Is Required for Autophagy in Mammalian Cells

Using a bioinformatic approach, we identified a TP53INP1-related gene encoding a protein with 30% identity with tumor protein 53-induced nuclear protein 1 (TP53INP1), which was named TP53INP2. TP53INP1 and TP53INP2 sequences were found in several species ranging from Homo sapiens to Drosophila melanogaster, but orthologues were found neither in earlier eukaryotes nor in prokaryotes. To gain insight into the function of the TP53INP2 protein, we carried out a yeast two-hybrid screening that showed that TP53INP2 binds to the LC3-related proteins GABARAP and GABARAP-like2, and then we demonstrated by coimmunoprecipitation that TP53INP2 interacts with these proteins, as well as with LC3 and with the autophagosome transmembrane protein VMP1. TP53INP2 translocates from the nucleus to the autophagosome structures after activation of autophagy by rapamycin or starvation. Also, we showed that TP53INP2 expression is necessary for autophagosome development because its small interfering RNA-mediated knockdown strongly decreases sensitivity of mammalian cells to autophagy. Finally, we found that interactions between TP53INP2 and LC3 or the LC3-related proteins GABARAP and GABARAP-like2 require autophagy and are modulated by wortmannin as judged by bioluminescence resonance energy transfer assays. We suggest that TP53INP2 is a scaffold protein that recruits LC3 and/or LC3-related proteins to the autophagosome membrane by interacting with the transmembrane protein VMP1. It is concluded that TP53INP2 is a novel gene involved in the autophagy of mammalian cells.     

Atg14L and Rubicon: Yin and yang of Beclin 1-mediated autophagy control

Emerging evidence suggests that Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, functions to coordinate two important cellular pathways: autophagy and apoptosis. Beclin 1 is a component of the Vps34/class III phosphatidylinositol 3-kinase (PtdIns3K) protein complex. However, the Beclin 1-Vps34/PtdIns3K protein complex formation and its function in autophagy regulation remain to be elucidated. Through an integrated approach that combines mouse genetics and biochemistry, we identified two novel Beclin 1 interacting proteins, Atg14L and Rubicon. We found that Atg14L and Rubicon play opposing roles in autophagy regulation by forming distinct complexes with Beclin 1, modulating the Vps34/PtdIns3K activity and targeting distinct steps of the autophagic process.     

A landmark protein essential for mitophagy

Degradation of mitochondria is a fundamental process conserved from yeast to humans that utilizes the machinery of autophagy. In contrast to starvation-induced, nonselective autophagy responsible for nutrient recycling, selective autophagy, which involves particular cues and receptors required for induction and cargo recognition, respectively, mediates mitochondria-specific breakdown. Although numerous studies highlight that mitochondria autophagy (mitophagy) contributes to homeostatic control of mitochondria, the molecular mechanisms underlying this selective clearance process are poorly understood. Using a genome-wide visual screen, we identified Atg32, a protein essential for mitophagy in budding yeast. During respiratory growth, Atg32 is highly expressed, likely in response to oxidative stress, and anchored on the surface of mitochondria. We also demonstrate that Atg32 interacts with Atg8 and Atg11, proteins critical for recognition of cargo receptors. Notably, Atg32 contains WXXI/L/V, a conserved motif that serves as a binding site for the Atg8 family members. Our recent findings suggest that Atg32 is a transmembrane receptor that directs autophagosome formation to mitochondria.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Δvps15 larvae. Fluorescence microscopy showed that Δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates.. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

A new class of ubiquitin-Atg8 receptors involved in selective autophagy and polyQ protein clearance

Selective ubiquitin-dependent autophagy mediates the disposal of superfluous cellular structures and clears cells of protein aggregates such as polyQ proteins linked to Huntington disease. Crucial selectivity factors of this pathway are ubiquitin-Atg8 receptors such as human SQSTM1/p62, which recognize ubiquitinated cargoes and deliver them to phagophores for degradation. Contrasting previous beliefs, we recently showed that ubiquitin-dependent autophagy is not restricted to higher eukaryotes but also exists in yeast with Cue5, harboring a ubiquitin-binding CUE domain, being a ubiquitin-Atg8 receptor. Notably, the human CUE domain protein TOLLIP is functionally similar to yeast Cue5, indicating that Cue5/TOLLIP (CUET) proteins represent a new and conserved class of autophagy receptors. Remarkably, both Cue5 in yeast and TOLLIP in human cells mediate efficient clearance of aggregation-prone polyQ proteins derived from human HTT/huntingtin. Indeed, TOLLIP is potentially more potent in polyQ clearance than SQSTM1/p62 in HeLa cells, suggesting that TOLLIP, also implicated in innate immunity, may be significant for human health and disease.     

A new class of ubiquitin-Atg8 receptors involved in selective autophagy and polyQ protein clearance

Selective ubiquitin-dependent autophagy mediates the disposal of superfluous cellular structures and clears cells of protein aggregates such as polyQ proteins linked to Huntington disease. Crucial selectivity factors of this pathway are ubiquitin-Atg8 receptors such as human SQSTM1/p62, which recognize ubiquitinated cargoes and deliver them to phagophores for degradation. Contrasting previous beliefs, we recently showed that ubiquitin-dependent autophagy is not restricted to higher eukaryotes but also exists in yeast with Cue5, harboring a ubiquitin-binding CUE domain, being a ubiquitin-Atg8 receptor. Notably, the human CUE domain protein TOLLIP is functionally similar to yeast Cue5, indicating that Cue5/TOLLIP (CUET) proteins represent a new and conserved class of autophagy receptors. Remarkably, both Cue5 in yeast and TOLLIP in human cells mediate efficient clearance of aggregation-prone polyQ proteins derived from human HTT/huntingtin. Indeed, TOLLIP is potentially more potent in polyQ clearance than SQSTM1/p62 in HeLa cells, suggesting that TOLLIP, also implicated in innate immunity, may be significant for human health and disease.     

New insights into autophagy using a multiple knockout strain

Not only is autophagy the major intracellular pathway for degradation and recycling of long-lived proteins and organelles, it is also involved in both the pathogenesis and prevention of many human diseases. Much progress has been made on the identification and characterization of AuTophaGy-related (ATG) genes, in yeast and in mammals. However, our understanding of the molecular mechanisms of autophagy remains quite limited, far from enough to harness autophagy for therapeutic applications. To better understand the molecular mechanisms, we took a unique and novel approach to study autophagy in yeast. We generated a multiple knockout Saccharomyces cerevisiae strain with 24 ATG genes deleted, and determined the minimum requirements for different aspects of autophagy. Our data also provided us with new insights into autophagy, different from those obtained from in vitro analyses. In this addendum, we briefly discuss our findings and consider fields where this multiple knockout strain can be of potential use.     

The coiled-coil domain protein EPG-8 plays an essential role in the autophagy pathway in C. elegans

Macroautophagy (hereafter referred to as autophagy) involves the formation of a closed, double membrane structure, called the autophagosome. Most of the Atg proteins that are essential for autophagosome formation are evolutionarily conserved between yeast and higher eukaryotes. The functions of some Atg proteins, however, are mediated by highly divergent proteins in mammalian cells. In this study, we identified a novel coiled-coil domain protein, EPG-8, that plays an essential role in the autophagy pathway in C. elegans. Mutations in epg-8 cause defects in degradation of various autophagy substrates and also compromise survival of animals under nutrient-depletion conditions. In epg-8 mutants, lipidated LGG-1 (the C. elegans Atg8 homolog) accumulates but does not form distinct punctate structures. EPG-8 directly interacts with the C. elegans Beclin 1 homolog, BEC-1. Our study demonstrates that epg-8 may function as a highly divergent homolog of the yeast autophagy gene Atg14.     

Unsaturated fatty acids induce non-canonical autophagy

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1^+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.     

Uncoupling proteins 1 and 3 are regulated differently

Using a heterologous yeast expression system, we have previously found a marked discordance between the effects of uncoupling protein (UCP) 1 and UCP3L on basal O(2) consumption in whole yeast versus isolated mitochondria. In whole yeast, UCP3L produces a greater stimulation of basal O(2) consumption, while in isolated mitochondria, UCP1 produces a much greater effect. As shown previously and in this report, UCP3L, in contrast to UCP1, is not inhibited by purine nucleotides. In the present study, we addressed two hypothetical mechanisms that could account for the observed discordance: (i) in whole yeast, purine nucleotides inhibit UCP1 but not UCP3L and (ii) preparations of isolated mitochondria lack an activator of UCP3L that is normally present in vivo. By use of a mutant of UCP1 that lacks purine nucleotide inhibition, it is demonstrated that cytosolic concentrations of purine nucleotides present in yeast effectively inhibit UCP1 activity. This suggests that the lower activity of UCP1 compared to UCP3L in whole yeast is due to purine nucleotide inhibition of UCP1 but not UCP3L. As potential activators of UCP3L we tested free fatty acids in whole yeast and isolated mitochondria. While UCP1 was strongly activated by free fatty acids, no stimulatory effect on UCP3L was observed. In summary, this study indicates that UCP1 and UCP3L differ in their regulation by purine nucleotides and free fatty acids. This different regulation may be related to different physiological functions of the two proteins.     

Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.

The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.     

Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two-hybrid screen in yeast and a co-purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non-homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.     

TRIM-directed selective autophagy regulates immune activation

Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.     

A fluorescent tool set for yeast Atg proteins

Fluorescence microscopy of live cells is instrumental in deciphering the molecular details of autophagy. To facilitate the routine examination of yeast Atg proteins under diverse conditions, here we provide a comprehensive tool set, including (1) plasmids for the expression of GFP chimeras at endogenous levels for most Atg proteins, (2) RFP-Atg8 constructs with improved properties as a PAS marker, and (3) plasmids for the complementation of common yeast auxotrophic markers. We hope that the availability of this tool set will further accelerate yeast autophagy research.     

Open and closed HORMAs regulate autophagy initiation

The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.     

Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.