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1.  Exit from the Golgi Is Required for the Expansion of the Autophagosomal Phagophore in Yeast Saccharomyces cerevisiae 
Molecular Biology of the Cell  2010;21(13):2270-2284.
The delivery of proteins and organelles to the vacuole by autophagy involves membrane rearrangements that result in the formation of autophagosomes. We have investigated the role of the Golgi in autophagy and found that, in yeast, this organelle plays a crucial role in supplying lipid bilayers necessary for autophagosome biogenesis.
The delivery of proteins and organelles to the vacuole by autophagy involves membrane rearrangements that result in the formation of large vesicles called autophagosomes. The mechanism underlying autophagosome biogenesis and the origin of the membranes composing these vesicles remains largely unclear. We have investigated the role of the Golgi complex in autophagy and have determined that in yeast, activation of ADP-ribosylation factor (Arf)1 and Arf2 GTPases by Sec7, Gea1, and Gea2 is essential for this catabolic process. The two main events catalyzed by these components, the biogenesis of COPI- and clathrin-coated vesicles, do not play a critical role in autophagy. Analysis of the sec7 strain under starvation conditions revealed that the autophagy machinery is correctly assembled and the precursor membrane cisterna of autophagosomes, the phagophore, is normally formed. However, the expansion of the phagophore into an autophagosome is severely impaired. Our data show that the Golgi complex plays a crucial role in supplying the lipid bilayers necessary for the biogenesis of double-membrane vesicles possibly through a new class of transport carriers or a new mechanism.
PMCID: PMC2893990  PMID: 20444982
2.  Atg27 Is Required for Autophagy-dependent Cycling of Atg9 
Molecular Biology of the Cell  2007;18(2):581-593.
Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.
PMCID: PMC1783788  PMID: 17135291
3.  The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis 
eLife  2013;2:e00947.
Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.
eLife digest
Cells continually adapt their behavior to accommodate changes in their environment. For example, when nutrients are abundant, cells can grow or proliferate; in times of scarcity, however, they must conserve resources for essential tasks. In particular, during periods of starvation, cells can cannibalize themselves in a process called autophagy, which literally means ‘self-eating’. Structures called autophagosomes engulf bits of cytoplasm and carry the contents to the digestive compartment of the cell, the lysosome, to be broken down into their constituent parts. This can include the degradation of proteins into amino acids, which can then be recycled into other proteins needed by the cell.
In cells, proteins are shipped to their destinations—which can be the plasma membrane or a specific organelle within the cell—via a delivery system known as the secretory pathway. This pathway begins in the endoplasmic reticulum (ER), where many of these proteins are made. From the ER, the proteins move to a compartment called the Golgi apparatus, which then sends them to their destinations, or to the lysosome to be broken down. Between the ER and Golgi they pass through a structure called the ER–Golgi intermediate compartment (ERGIC).
Although the signaling pathways that initiate autophagy are known, less is understood about the actual formation of the autophagosomes. Now, Ge et al. have developed an in vitro system to study their formation, and gone on to identify a membrane that is both necessary and sufficient to create these structures.
Previous studies have implicated a variety of membranes—including the plasma membrane and the membranes belonging to the ER, the Golgi apparatus, the lysosome and various other organelles—in the formation of autophagosomes. To identify which of these membranes might be involved, Ge et al. focused on a protein called LC3 that is a key marker for the formation of the autophagosome. This protein is recruited to the growing autophagosome by a lipid, so discovering which membranes can add a lipid to LC3 should shed light on the assembly process.
By separating the full range of organelles in a cell lysate into fractions (a process called fractionation), Ge et al. found that the ERGIC was the most active membrane to attach lipid to LC3. Additionally, the lipid was only added when signaling pathways that stimulate autophagy—such as the PI3K pathway—were activated. Together, these results provide insight into the mechanism of autophagosome formation, and the structures in the cell that participate in this process.
PMCID: PMC3736544  PMID: 23930225
autophagy; ER–Golgi intermediate compartment; LC3 lipidation; autophagosome; Human; Mouse
4.  Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy 
Cell & Bioscience  2014;4:16.
Autophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes.
Using electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy.
With the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome.
PMCID: PMC3977894  PMID: 24669863
Breast cancer; Mitochondria; Autophagy; Mitophagy; Parkin; Antiestrogen resistance; Fulvestrant; Imatinib; Estrogen receptor-α
5.  The Golgi as a potential membrane source for autophagy 
Autophagy  2010;6(7):950-951.
In macroautophagy (hereafter autophagy), a morphological hallmark is the formation of double-membrane vesicles called autophagosomes that sequester and deliver cytoplasmic components to the lysosome/vacuole for degradation. This process begins with an initial sequestering compartment, the phagophore, which expands into the mature autophagosome. A tremendous amount of work has been carried out to elucidate the mechanism of how the autophagosome is formed. However, an important missing piece in this puzzle is where the membrane comes from. Independent lines of evidence have shown that preexisting organelles may continuously supply lipids to support autophagosome formation. In our analysis, we identified several components of the late stage secretory pathway that may redirect Golgi-derived membrane to autophagosome formation in response to starvation conditions.
PMCID: PMC3359472  PMID: 20729630
lysosome; membrane biogenesis; protein targeting; secretory pathway; stress; vacuole; yeast
6.  A current perspective of autophagosome biogenesis 
Cell Research  2013;24(1):58-68.
Autophagy is a bulk degradation system induced by cellular stresses such as nutrient starvation. Its function relies on the formation of double-membrane vesicles called autophagosomes. Unlike other organelles that appear to stably exist in the cell, autophagosomes are formed on demand, and once their formation is initiated, it proceeds surprisingly rapidly. How and where this dynamic autophagosome formation takes place has been a long-standing question, but the discovery of Atg proteins in the 1990's significantly accelerated our understanding of autophagosome biogenesis. In this review, we will briefly introduce each Atg functional unit in relation to autophagosome biogenesis, and then discuss the origin of the autophagosomal membrane with an introduction to selected recent studies addressing this problem.
PMCID: PMC3879706  PMID: 24296784
autophagy; autophagosome; Atg
7.  Mitochondria supply membranes for autophagosome biogenesis during starvation 
Cell  2010;141(4):656-667.
Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby re-supplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, autophagosomes are derived from the outer membranes of mitochondria. In time-lapse movies, the early autophagosomal marker, mApg5, transiently localizes to punctae on the surface of mitochondria, followed by the late autophagosomal marker, LC3. A unique tail-anchored outer mitochondrial membrane protein, but not other outer nor inner mitochondrial membrane proteins, labels autophagosomes and diffuses into newly forming autophagosomes from mitochondria. The fluorescent lipid, NBD-PS (which converts to PE in mitochondria) transfers from mitochondria to autophagosomes in starved cells. In addition, when mitochondria/ER connections are perturbed by loss of mitofusin2, starvation-induced autophagosomes do not form. Mitochondria thus play a central role in starvation-induced autophagy, serving as membrane source of autophagosomes.
PMCID: PMC3059894  PMID: 20478256
8.  Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites 
PLoS Pathogens  2011;7(12):e1002416.
Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
Author Summary
Autophagy is a catabolic process involved in maintaining cellular homeostasis in eukaryotic cells, while coping with their changing environmental conditions. Mechanistically, it is also a process of considerable complexity involving multiple protein factors and implying numerous protein-protein and protein-membrane interactions. The cellular material to be degraded by autophagy is contained in a membrane-bound compartment called the autophagosome. We have characterised the formation of autophagosomes in the protozoan parasite Toxoplasma gondii by following the relocalisation of autophagosome-bound TgAtg8. Thus, exploiting GFP-TgAtg8 as a marker, we showed that it is a process that is regulated and can be induced artificially by amino acid starvation. Autophagic vesicles were also observed in normally dividing intracellular parasites. Depleting Toxoplasma of the TgAtg3 autophagy protein led to an impairment of TgAtg8 conjugation to the autophagosomal membrane and, at the cellular level, to a fragmentation of the single mitochondrion of the parasite and to a severe growth arrest. We have thus found that TgAtg3-dependent autophagy is essential for normal intracellular development of T. gondii tachyzoites.
PMCID: PMC3228817  PMID: 22144900
9.  Post-Golgi Sec Proteins Are Required for Autophagy in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2010;21(13):2257-2269.
Macroautophagy is linked to various diseases in humans, including cancer and neurodegeneration. The morphological hallmark is the formation of the double-membrane autophagosome, which is the most complex aspect of macroautophagy. We demonstrate a role for post-Golgi Sec proteins, Sec2 and Sec4, in autophagosome formation.
In eukaryotic cells, autophagy mediates the degradation of cytosolic contents in response to environmental change. Genetic analyses in fungi have identified over 30 autophagy-related (ATG) genes and provide substantial insight into the molecular mechanism of this process. However, one essential issue that has not been resolved is the origin of the lipids that form the autophagosome, the sequestering vesicle that is critical for autophagy. Here, we report that two post-Golgi proteins, Sec2 and Sec4, are required for autophagy. Sec4 is a Rab family GTPase, and Sec2 is its guanine nucleotide exchange factor. In sec2 and sec4 conditional mutant yeast, the anterograde movement of Atg9, a proposed membrane carrier, is impaired during starvation conditions. Similarly, in the sec2 mutant, Atg8 is inefficiently recruited to the phagophore assembly site, which is involved in autophagosome biogenesis, resulting in the generation of fewer autophagosomes. We propose that following autophagy induction the function of Sec2 and Sec4 are diverted to direct membrane flow to autophagosome formation.
PMCID: PMC2893989  PMID: 20444978
10.  Autophagy: More Than a Nonselective Pathway 
Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs). For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy.
PMCID: PMC3362037  PMID: 22666256
11.  ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major 
PLoS Pathogens  2012;8(5):e1002695.
Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.
Author Summary
Leishmaniasis is a disease of humans that is of major significance throughout many parts of the world. It is caused by the protozoan parasite Leishmania and mammals are infected through the bite of a sand fly in which the parasite develops. Parasite remodelling crucial for generation of the human-infective forms is aided by the catabolic process known as autophagy in which cell material is packaged within organelles called autophagosomes and subsequently broken down in the digestive lysosomal compartment. Here we show that autophagy in Leishmania requires the coordinated actions of two pathways, one of which involves a protein called ATG5. We have generated parasite mutants lacking this protein and shown that ATG5 is required for both autophagosome formation and also maintenance of a fully functional mitochondrion. The mutants lacking ATG5 have increased mitochondrial mass and phospholipid content, high levels of oxidants and reduced membrane potential, all being hallmarks of a dysfunctional mitochondrion with impaired ability for energy generation. Our results have thus revealed that a functional autophagic pathway is crucial for phospholipid homeostasis and mitochondrial function in the parasite and important for the parasite's differentiation, infectivity and virulence to its mammalian host.
PMCID: PMC3355087  PMID: 22615560
12.  Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D 
The Journal of Cell Biology  2012;196(4):483-496.
Arf6 positively regulates autophagosome membrane biogenesis by inducing PIP2 generation and PLD activation, which together may influence endocytic uptake of plasma membrane into autophagosome precursors.
Macroautophagy (in this paper referred to as autophagy) and the ubiquitin–proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane–bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP2) and in inducing phospholipase D (PLD) activity. PIP2 and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.
PMCID: PMC3283994  PMID: 22351926
13.  The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy 
The Journal of Cell Biology  2010;188(1):101-114.
COG subunits localize to the phagophore assembly site where they interact with autophagy proteins and are required for double-membrane Cvt vesicle and autophagosome formation.
Macroautophagy is a catabolic pathway used for the turnover of long-lived proteins and organelles in eukaryotic cells. The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm. Autophagosome formation is the most complex part of macroautophagy, and it is a dynamic event that likely involves vesicle fusion to expand the initial sequestering membrane, the phagophore; however, essentially nothing is known about this process including the molecular components involved in vesicle tethering and fusion. In this study, we provide evidence that the subunits of the conserved oligomeric Golgi (COG) complex are required for double-membrane cytoplasm to vacuole targeting vesicle and autophagosome formation. COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins. In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.
PMCID: PMC2812853  PMID: 20065092
14.  Mammalian autophagy: core molecular machinery and signaling regulation 
Current opinion in cell biology  2009;22(2):124-131.
Autophagy, a cellular catabolic pathway, is evolutionarily conserved from yeast to mammals. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering long-lived proteins and excess or damaged organelle into the lysosome for degradation and reuse of the resulting macromolecules. In addition to the hallmark discovery of core molecular machinery components involved in autophagosome formation, complex signaling cascades controlling autophagy have also begun to emerge, with mTOR as a central but far from exclusive player. Malfunction of autophagy has been linked to a wide range of human pathologies, including cancer, neurodegeneration and pathogen infection. Here we highlight recent advances in identifying and understanding the core molecular machinery and signaling pathways that are involved in mammalian autophagy.
PMCID: PMC2854249  PMID: 20034776
autophagy; lysosomes; mammalian cells; signal transduction; stress
15.  Diverse Autophagosome Membrane Sources Coalesce in Recycling Endosomes 
Cell  2013;154(6):1285-1299.
Autophagic protein degradation is mediated by autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.
Graphical Abstract
•mATG9 traffics from the plasma membrane to recycling endosomes•mATG9 vesicles fuse with ATG16L1 vesicles in recycling endosomes•VAMP3, Rab11, myosin Vb, and starvation regulate mATG9-ATG16L1 vesicle fusion•mATG9-ATG16L1 vesicle fusions regulate autophagosome formation
Autophagosome membranes that originate in different cellular compartments follow distinct routes to recycling endosomes, where they fuse to form autophagosome precursors.
PMCID: PMC3791395  PMID: 24034251
16.  Autophagosome Precursor Maturation Requires Homotypic Fusion 
Cell  2011;146(2):303-317.
Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.
Graphical Abstract
► Homotypic fusion of Atg16L1 vesicles enables their maturation into autophagosomes ► VAMP7 regulates Atg16L1 vesicle homotypic fusion and autophagosome formation ► Plasma membrane is a likely source of SNAREs required for Atg16L1 vesicle fusion ► Starvation, which induces autophagy, triggers Atg16L1 vesicle homotypic fusion
PMCID: PMC3171170  PMID: 21784250
17.  Infection with Usutu Virus Induces an Autophagic Response in Mammalian Cells 
Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection.
Author Summary
The identification of cellular components and metabolic pathways involved in virus replication provides valuable information for the development of new antiviral strategies. Autophagy is one of these metabolic pathways with multiple implications during viral replication. Autophagy literally means self-digestion and constitutes a cellular process by which intracellular components are enclosed by membrane structures and degraded. Interestingly autophagy can contribute either positively or negatively to viral infections. For instance, several viruses hijack these autophagic membranes to build their replication complexes or take advantage on metabolic rearrangements induced following autophagy, while in other cases autophagy contributes to viral clearance and innate immunity. In this study, we explored the possible implication of the autophagic pathway during Usutu virus infection (USUV). USUV is an African mosquito-borne flavivirus that mainly infects mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Our results indicate that infection by USUV of different origins triggers an autophagic response within infected cells. Even more, drugs that target components from the autophagic pathway modulate USUV-infection. These results provide the basis for the design of new antiviral research lines against this pathogen.
PMCID: PMC3812092  PMID: 24205422
18.  Multiple roles of the cytoskeleton in autophagy 
Autophagy is involved in a wide range of physiological processes including cellular remodeling during development, immuno-protection against heterologous invaders and elimination of aberrant or obsolete cellular structures. This conserved degradation pathway also plays a key role in maintaining intracellular nutritional homeostasis and during starvation, for example, it is involved in the recycling of unnecessary cellular components to compensate for the limitation of nutrients. Autophagy is characterized by specific membrane rearrangements that culminate with the formation of large cytosolic double-membrane vesicles called autophagosomes. Autophagosomes sequester cytoplasmic material that is destined for degradation. Once completed, these vesicles dock and fuse with endosomes and/or lysosomes to deliver their contents into the hydrolytically active lumen of the latter organelle where, together with their cargoes, they are broken down into their basic components. Specific structures destined for degradation via autophagy are in many cases selectively targeted and sequestered into autophagosomes.
A number of factors required for autophagy have been identified, but numerous questions about the molecular mechanism of this pathway remain unanswered. For instance, it is unclear how membranes are recruited and assembled into autophagosomes. In addition, once completed, these vesicles are transported to cellular locations where endosomes and lysosomes are concentrated. The mechanism employed for this directed movement is not well understood. The cellular cytoskeleton is a large, highly dynamic cellular scaffold that has a crucial role in multiple processes, several of which involve membrane rearrangements and vesicle-mediated events. Relatively little is known about the roles of the cytoskeleton network in autophagy. Nevertheless, some recent studies have revealed the importance of cytoskeletal elements such as actin microfilaments and microtubules in specific aspects of autophagy. In this review, we will highlight the results of this work and discuss their implications, providing possible working models. In particular, we will first describe the findings obtained with the yeast Saccharomyces cerevisiae, for long the leading organism for the study of autophagy, and, successively, those attained in mammalian cells, to emphasize possible differences between eukaryotic organisms.
PMCID: PMC2831541  PMID: 19659885
autophagy; autophagosome; Cvt pathway; actin; microtubules; cytoskeleton
19.  Loss of a membrane trafficking protein αSNAP induces non-canonical autophagy in human epithelia 
Cell Cycle  2012;11(24):4613-4625.
Autophagy is a catabolic process that sequesters intracellular proteins and organelles within membrane vesicles called autophagosomes with their subsequent delivery to lyzosomes for degradation. This process involves multiple fusions of autophagosomal membranes with different vesicular compartments; however, the role of vesicle fusion in autophagosomal biogenesis remains poorly understood. This study addresses the role of a key vesicle fusion regulator, soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP), in autophagy. Small interfering RNA-mediated downregulation of αSNAP expression in cultured epithelial cells stimulated the autophagic flux, which was manifested by increased conjugation of microtubule-associated protein light chain 3 (LC3-II) and accumulation of LC3-positive autophagosomes. This enhanced autophagy developed via a non-canonical mechanism that did not require beclin1-p150-dependent nucleation, but involved Atg5 and Atg7-mediated elongation of autophagosomal membranes. Induction of autophagy in αSNAP-depleted cells was accompanied by decreased mTOR signaling but appeared to be independent of αSNAP-binding partners, N-ethylmaleimide-sensitive factor and BNIP1. Loss of αSNAP caused fragmentation of the Golgi and downregulation of the Golgi-specific GTP exchange factors, GBF1, BIG1 and BIG2. Pharmacological disruption of the Golgi and genetic inhibition of GBF1 recreated the effects of αSNAP depletion on the autophagic flux. Our study revealed a novel role for αSNAP as a negative regulator of autophagy that acts by enhancing mTOR signaling and regulating the integrity of the Golgi complex.
PMCID: PMC3562306  PMID: 23187805
Beclin1; NSF; mTOR; Bif-1; vesicle trafficking; Golgi disruption
20.  Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy 
Molecular Biology of the Cell  2012;23(10):1860-1873.
Mammalian Atg9 (mAtg9) is a multispanning membrane protein that resides in a novel compartment. mAtg9 interacts dynamically with phagophores and forming autophagosomes. It is proposed that mAtg9 function is required to initiate autophagosome formation and increase the number of autophagosomes.
Autophagy is a catabolic process essential for cell homeostasis, at the core of which is the formation of double-membrane organelles called autophagosomes. Atg9 is the only known transmembrane protein required for autophagy and is proposed to deliver membrane to the preautophagosome structures and autophagosomes. We show here that mammalian Atg9 (mAtg9) is required for the formation of DFCP1-positive autophagosome precursors called phagophores. mAtg9 is recruited to phagophores independent of early autophagy proteins, such as ULK1 and WIPI2, but does not become a stable component of the autophagosome membrane. In fact, mAtg9-positive structures interact dynamically with phagophores and autophagosomes without being incorporated into them. The membrane compartment enriched in mAtg9 displays a unique sedimentation profile, which is unaltered upon starvation-induced autophagy. Correlative light electron microscopy reveals that mAtg9 is present on tubular–vesicular membranes emanating from vacuolar structures. We show that mAtg9 resides in a unique endosomal-like compartment and on endosomes, including recycling endosomes, where it interacts with the transferrin receptor. We propose that mAtg9 trafficking through multiple organelles, including recycling endosomes, is essential for the initiation and progression of autophagy; however, rather than acting as a structural component of the autophagosome, it is required for the expansion of the autophagosome precursor.
PMCID: PMC3350551  PMID: 22456507
21.  Plasma membrane contributes to the formation of pre-autophagosomal structures 
Nature cell biology  2010;12(8):747-757.
Autophagy is a catabolic process where lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by elongation and fusion of phagophores, which derive from pre-autophagosomal structures. The membrane origins of autophagosomes are unclear and may involve multiple sources, including the endoplasmic reticulum and mitochondria. Here we show in mammalian cells that clathrin heavy-chain interacts with Atg16L1, and is involved in the formation of Atg16L1-positive early autophagosome precursors. Inhibition of clathrin-mediated internalisation reduced the formation of both Atg16L1-positive precursors and mature autophagosomes, while Atg16L1 associated with clathrin-coated structures. We tested and demonstrated that the plasma membrane (PM) directly contributes to the formation of early Atg16L1-positive autophagosome precursors. This may be particularly important during periods of increased autophagosome formation, as the plasma membrane may serve as a large membrane reservoir that allows cells periods of autophagosome synthesis at levels many-fold higher than under basal conditions, without compromising other processes.
PMCID: PMC2923063  PMID: 20639872
22.  Dysregulation of Autophagy in Murine Fibroblasts Resistant to HSV-1 Infection 
PLoS ONE  2012;7(8):e42636.
The mouse L cell mutant, gro29, was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1). gro29 cells are fully susceptible to HSV-1 infection, however, they produce 2000-fold less infectious virus than parental L cells despite their capacity to synthesize late viral gene products and assemble virions. Because productive HSV-1 infection is presumed to result in the death of the host cell, we questioned how gro29 cells might survive infection. Using time-lapse video microscopy, we demonstrated that a fraction of infected gro29 cells survived infection and divided. Electron microscopy of infected gro29 cells, revealed large membranous vesicles that contained virions as well as cytoplasmic constituents. These structures were reminiscent of autophagosomes. Autophagy is an ancient cellular process that, under nutrient deprivation conditions, results in the degradation and catabolism of cytoplasmic components and organelles. We hypothesized that enhanced autophagy, and resultant degradation of virions, might explain the ability of gro29 to survive HSV-1 infection. Here we demonstrate that gro29 cells have enhanced basal autophagy as compared to parental L cells. Moreover, treatment of gro29 cells with 3-methyladenine, an inhibitor of autophagy, failed to prevent the formation of autophagosome-like organelles in gro29 cells indicating that autophagy was dysregulated in these cells. Additionally, we observed robust co-localization of the virion structural component, VP26, with the autophagosomal marker, GFP-LC3, in infected gro29 cells that was not seen in infected parental L cells. Collectively, these data support a model whereby gro29 cells prevent the release of infectious virus by directing intracellular virions to an autophagosome-like compartment. Importantly, induction of autophagy in parental L cells did not prevent HSV-1 production, indicating that the relationship between autophagy, virus replication, and survival of HSV-1 infection by gro29 cells is complex.
PMCID: PMC3416809  PMID: 22900036
23.  Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae 
PLoS ONE  2014;9(3):e91651.
Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.
PMCID: PMC3953483  PMID: 24626240
24.  When autophagy meets viruses: a double-edged sword with functions in defense and offense 
Seminars in immunopathology  2010;32(4):323-341.
Autophagy is a ubiquitous catabolic process that ensures organism’s well-being by sequestering a wide array of undesired intracellular constituents into double-membrane vesicles termed autophagosomes for lysosomal degradation. Interest in autophagy research has recently gained momentum as it is increasingly being recognized to play fundamental roles in diverse aspects of human pathophysiology including virus infection and its subsequent complications. This review discusses recent advances in autophagy studies with respect to virus infection and pathogenesis. A growing body of evidence suggests that the autophagy pathway and/or autophagy genes play pleiotropic functions in the host’s intrinsic, innate, and adaptive immune response against viruses. However, some viruses have evolved to encode virulence factors that evade or counteract the execution of autophagy. Furthermore, certain viruses are equipped to enhance autophagy or exploit the autophagy machinery for their replication and pathogenesis. A comprehensive understanding of the roles of autophagy pathway and autophagy genes during viral infection may enable the discovery of novel antiviral drug targets.
PMCID: PMC3169181  PMID: 20865416
Autophagy; Antiviral host defense; Viral infection; Viral replication; Viral pathogenesis
25.  Autophagy in mammalian cells 
Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The pre-autophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5’-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an important cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.
PMCID: PMC3272585  PMID: 22312452
Autophagy; Phagophore; Autophagosome; Atg proteins; Cell survival

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