The intersection of Golgi-ER retrograde and autophagic trafficking

For decades, a marvelous amount of work has been performed to identify molecules that regulate distinct stages of membrane transport in the ER-Golgi secretory pathway and autophagy, which are implicated in many human diseases. However, an important missing piece in this puzzle is how the cell dynamically coordinates these crisscrossed trafficking pathways in response to different stimuli. Our recent study has identified UVRAG as a mode-switching protein that coordinates Golgi-ER retrograde and autophagic trafficking. UVRAG recognizes phosphatidylinositol-3-phosphate (PtdIns3P) and locates to the ER, where it couples the ER tethering complex containing RINT1 to govern Golgi-ER retrograde transport. Intriguingly, when autophagy is induced, UVRAG undergoes a “partnering shift” from the ER tethering complex to the BECN1 autophagy complex, resulting in concomitant inhibition of Golgi-ER transport and the activation of ATG9 autophagic trafficking. Therefore, Golgi-ER retrograde and autophagy-related membrane trafficking are functionally interdependent and tightly regulated by UVRAG to ensure spatiotemporal fidelity of protein transport and organelle homeostasis, providing distinguished insights into trafficking-related diseases.     

Beyond autophagy: the role of UVRAG in membrane trafficking

Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.     

Beyond autophagy: The role of UVRAG in membrane trafficking

Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.

Addendum to: Liang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, Vergne I, Deretic V, Feng P, Akazawa C, Jung JU. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol 2008; 10:776–87.     

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

UVRAG mutations associated with microsatellite unstable colon cancer do not affect autophagy

Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI ) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HE K cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.     

Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein are required for the stability of the Q/t-SNARE complex at the endoplasmic reticulum in yeast

The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi-ER retrograde transport. Size exclusion chromatography and affinity purification approaches confirmed that Dsl3p is associated with subunits of the "Dsl1p complex." The complex also includes the Q/t-SNARE proteins, Use1p, Sec20p, and Ufe1p, integral membrane proteins that constitute the trimeric acceptor for R/v-SNAREs on Golgi-derived vesicles at the ER. Using mutants, we performed a detailed analysis of interactions between subunits of the Dsl1p complex and the ER-localized SNARE proteins. This analysis showed that both Dsl1p and Dsl3p are required for the stable interaction of the SNARE Use1p with a central subcomplex consisting of Tip20p and the SNARE proteins Ufe1p and Sec20p.     

Retrograde trafficking from the vacuole/lysosome membrane

Membrane protein recycling is a fundamental process from yeast to humans. The lysosome (or vacuole in yeast) receives membrane proteins from the secretory, endocytic, and macroautophagy/autophagy pathways. Although some of these membrane proteins appear to be recycled, the molecular mechanisms underlying this retrograde trafficking are poorly understood. Our recent study revealed that the transmembrane autophagy protein Atg27 is recycled from the vacuole membrane using a 2-step recycling process. First, the Snx4 complex recycles Atg27 from the vacuole to the endosome. Then, the retromer complex mediates endosome-to-Golgi retrograde transport. Thus, 2 distinct protein complexes facilitate the sequential retrograde trafficking for Atg27. As far as we know, Atg27 is the first physiological substrate for the vacuole-to-endosome retrograde trafficking pathway.     

ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis

The double‐membrane‐bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl‐inositol‐3‐phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E‐Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E‐Syt‐containing domains during autophagy and that inhibition of E‐Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E‐Syts are essential for autophagy‐associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.     

Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum

Insights into the function of the Golgi complex have been provided by experiments performed with various inhibitors of membrane trafficking, such as the macrocyclic lactone brefeldin A (BFA), a compound that inhibits constitutive secretion, prevents the formation of coatomer-coated transport vesicles, and stimulates the retrograde movement of Golgi resident enzymes back to the ER. We show here that the structurally unrelated compound clofibrate, a peroxisome proliferator (PP) and hypolipidemic agent, also reversibly disrupts the morphological and functional integrity of the Golgi complex in a manner similar to BFA. In the presence of clofibrate, the forward transport of newly synthesized secretory proteins from the ER to the Golgi is dramatically inhibited. Moreover, clofibrate causes Golgi membranes to travel rapidly in a microtubule-dependent manner back to the ER, forming a hybrid ER–Golgi tubulovesicular membrane network. These affects appear to be independent of clofibrate's ability to stimulate the PP-activated receptor (PPAR) alpha pathway because other PPAR stimulators (DEHP, WY-14643) did not alter the Golgi complex or induce retrograde trafficking. These data suggest that PPAR alpha-independent, clofibrate-sensitive proteins participate in regulating Golgi-to-ER retrograde membrane transport, and, equally importantly, that clofibrate may be used as a pharmacological tool for investigating Golgi membrane dynamics.     

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

Autophagy in yeast: a review of the molecular machinery

Autophagy is a membrane trafficking mechanism that delivers cytoplasmic cargo to the vacuole/lysosome for degradation and recycling. In addition to non-specific bulk cytosol, selective cargoes, such as peroxisomes, are sorted for autophagic transport under specific physiological conditions. In a nutrient-rich growth environment, many of the autophagic components are recruited for executing a biosynthetic trafficking process, the cytoplasm to vacuole targeting (Cvt) pathway, that transports the resident hydrolases aminopeptidase I and alpha-mannosidase to the vacuole in Saccharomyces cerevisiae. Recent studies have identified pathway-specific components that are necessary to divert a protein kinase and a lipid kinase complex to regulate the conversion between the Cvt pathway and autophagy. Downstream of these proteins, the general machinery for transport vesicle formation involves two novel conjugation systems and a putative membrane protein complex. Completed vesicles are targeted to, and fuse with, the vacuole under the control of machinery shared with other vacuolar trafficking pathways. Inside the vacuole, a potential lipase and several proteases are responsible for the final steps of vesicle breakdown, precursor enzyme processing and substrate turnover. In this review, we discuss the most recent developments in yeast autophagy and point out the challenges we face in the future.     

Roles of Pichia pastoris Uvrag in vacuolar protein sorting and the phosphatidylinositol 3-kinase complex in phagophore elongation in autophagy pathways

Although it has been established that Atg6/Beclin 1, the phosphatidylinositol 3-kinase (PI3K) Vps34, and associated proteins have direct or indirect roles in autophagic pathways in both mammals and yeasts, the elucidation of these roles and the proteins required for them is ongoing. The involvement of the Beclin 1-binding protein, UVRAG, has been a particular source of disagreement. We found that PpAtg6 is required for all autophagic pathways that have been identified in the yeast Pichia pastoris, as well as for the carboxypeptidase Y (PpCPY) vacuolar protein sorting pathway. We localized PpAtg6 to the phagophore assembly site (PAS) and observed its continued presence at that site as the isolation membrane grew from it and matured into a pexophagosome. PpUvrag, however, was required for proper PpCPY sorting, but not for any autophagic pathway. Rather, the defects in all autophagic pathways observed when PpUvrag was overexpressed support its presence in a complex that competes with the PI3K complex required for autophagy.     

The autophagic roles of Rab small GTPases and their upstream regulators: A review

Macroautophagy is an evolutionarily conserved degradative process of eukaryotic cells. Double-membrane vesicles called autophagosomes sequester portions of cytoplasm and undergo fusion with the endolysosomal pathway in order to degrade their content. There is growing evidence that members of the small GTPase RAB protein family—the well-known regulators of membrane trafficking and fusion events—play key roles in the regulation of the autophagic process. Despite numerous studies focusing on the functions of RAB proteins in autophagy, the importance of their upstream regulators in this process emerged only in the past few years. In this review, we summarize recent advances on the effects of RABs and their upstream modulators in the regulation of autophagy. Moreover, we discuss how impairment of these proteins alters the autophagic process leading to several generally known human diseases.     

Control of Golgi morphology and function by Sed5 t-SNARE phosphorylation

Previously, we demonstrated that the phosphorylation of t-SNAREs by protein kinase A (PKA) affects their ability to participate in SNARE complexes and to confer endocytosis and exocytosis in yeast. Here, we show that the presumed phosphorylation of a conserved membrane-proximal PKA consensus site (serine-317) in the Sed5 t-SNARE regulates endoplasmic reticulum (ER)-Golgi transport, as well as Golgi morphology. Sed5 is a phosphoprotein, and both alanine and aspartate substitutions in serine-317 directly affect intracellular protein trafficking. The aspartate substitution results in elaboration of the ER, defects in Golgi-ER retrograde transport, an accumulation of small transport vesicles, and the inhibition of growth of most cell types. In contrast, the alanine substitution has no deleterious effects upon transport and growth, but results in ordering of the Golgi into a structure reminiscent of mammalian apparatus. This structure seems to require the recycling of Sed5, because it was found not to occur in sec21-2 cells that are defective in retrograde transport. Thus, a cycle of Sed5 phosphorylation and dephosphorylation is required for normal t-SNARE function and may choreograph Golgi ordering and dispersal.     

Quantitation of “autophagic flux” in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure “autophagic flux” in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the “autophagometer.” In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. the addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a “colchicine block.” Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an “in vivo autophagometer” study using colchicine in skeletal muscle.Key words: autophagy, rapamycin, skeletal muscle