The Golgi as a potential membrane source for autophagy

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 pre-existing 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.     

Mechanisms and regulation of autophagosome formation

Autophagy is an intracellular pathway for the bulk degradation of cytoplasmic substances such as cytosol, protein aggregates and organelles. Autophagy is characterized by the formation of double-membrane bound vesicles called autophagosomes, which engulf the cargo and transport it to the vacuole/lysosome for breakdown and recycling. Even though several proteins in this pathway have been identified, little is known about the mechanism of action of these proteins during autophagosome biogenesis. In this review we briefly discuss recent findings on the molecular players and mechanisms involved in autophagosome formation. In particular, we will focus on the mechanisms regulating membrane recruitment as well as membrane remodeling during autophagosome formation.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.     

Atg27 is required for autophagy-dependent cycling of Atg9

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.     

Mitochondria: One of the origins for autophagosomal membranes?

Macroautophagy is a transport pathway to the lysosome/vacuole that contributes to the degradation of numerous intracellular components. Despite the recent advances achieved in the understanding of the molecular mechanism underlying macroautophagy, the membrane origin of autophagosomes, the hallmark of this process is still a mystery. It has been suggested that mitochondria may be one of the lipid sources for autophagosome formation and that possibly this organelle provides the phosphatidylethanolamine (PE) that covalently links to the members of the ubiquitin-like Atg8/microtubule-associated protein 1 light chain 3 (LC3) protein family. These lipidated proteins are inserted into the outer and inner surface of autophagosomes and are essential for the biogenesis of these large double-membrane vesicles. However, because PE is an integral component of all cellular membranes, designing appropriate experiments to determine the origin of the autophagosomal PE is not easy. In this review, we discuss the idea that mitochondria provide the pool of PE necessary for the autophagosome biogenesis and we propose some possible experimental approaches aimed to explore this possibility.     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).     

LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis

Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana

Vacuolar autophagy is a major pathway by which eukaryotic cells degrade macromolecules, either to remove damaged or unnecessary proteins, or to produce respiratory substrates and raw materials to survive periods of nutrient deficiency. During autophagy, a double membrane forms around cytoplasmic components to generate an autophagosome, which is transported to the vacuole. The outer membrane fuses with the vacuole or lysosome, and the inner membrane and its contents are degraded by vacuolar or lysosomal hydrolases. We have identified a small gene family in Arabidopsis thaliana, members of which show sequence similarity to the yeast autophagy gene ATG18. Members of the AtATG18 gene family are differentially expressed in response to different growth conditions, and one member of this family, AtATG18a, is induced both during sucrose and nitrogen starvation and during senescence. RNA interference was used to generate transgenic lines with reduced AtATG18a expression. These lines show hypersensitivity to sucrose and nitrogen starvation and premature senescence, both during natural senescence of leaves and in a detached leaf assay. Staining with the autophagosome-specific fluorescent dye monodansylcadaverine revealed that, unlike wild-type plants, AtATG18a RNA interference plants are unable to produce autophagosomes in response to starvation or senescence conditions. We conclude that the AtATG18a protein is likely to be required for autophagosome formation in Arabidopsis.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

SNARE proteins are required for macroautophagy

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.     

Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways

To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.     

Autophagic degradation of leaf starch in plants

Autophagy is an evolutionarily conserved process in eukaryotic cells that functions to degrade cytoplasmic components in the vacuole or lysosome. Previous research indicates that the core molecular machinery of autophagosome formation works well in plants, and plant autophagy plays roles in diverse biological processes such as nutrient recycling, development, immunity and responses to a variety of abiotic stresses. Recently, we reported that autophagy contributed to leaf starch degradation, which had been thought to be a process confined to chloroplasts. This finding demonstrated a previously unidentified pathway of leaf starch depletion and a new role of basal autophagy in plants.     

The early secretory pathway contributes to autophagy in yeast

Autophagy is a starvation response in eukaryotes by which the cell delivers cytoplasmic components to the vacuole for degradation, and is mediated by a double membrane structure called the autophagosome. We have previously proposed that the specific combination of COPII like components, including Sec24p, is required for autophagy (Ishihara, N. et al. (2001) Mol. Biol. Cell, 12: 3690-3702). The autophagic defect in sec24 deleted mutant cells was, however, suppressed upon the recovery of its secretory flow by the overexpression of its homologue, Sfb2p. We have also reported that the autophagic defect is not observed in sec13 and sec31 mutants, a phenomenon that can be explained by the fact that starvation stress suppresses the secretory defect of these mutants. These observations indicate that the active flow in the early secretory pathway plays an important role in autophagy; that is, autophagy proceeds in the presence, but not in the absence of the early secretory flow. Both autophagy and its closely related cytoplasm to vacuole-targeting (Cvt) pathway occur through a pre-autophagosomal structure (PAS), and since the PAS and the functional Cvt pathway exist in all sec mutants, the early secretory pathway must be involved specifically in autophagy, subsequent to PAS formation.     

Vacuole Biogenesis in Saccharomyces cerevisiae: Protein Transport Pathways to the Yeast Vacuole

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.     

Characterization of a novel autophagy-specific gene, ATG29

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Delta cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.     

自噬过程的晚期阶段

自噬是高度保守的细胞内降解途径。在此过程中,部分细胞质和细胞器被双层膜的囊泡包裹形成自噬体,随后与溶酶体融合并降解被吞噬的物质。降解产物被释放到细胞质中重新用于必需的物质和能量合成。本文主要关注自噬的晚期阶段,即从自噬体合成结束到溶酶体再生过程。通过对这一过程相关基因及蛋白产物的研究,初步揭示了此过程的分子机制。     

Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy

Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II⁺ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP‐23 and SNAP‐29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.