ER-phagy: selective autophagy of the endoplasmic reticulum

Throughout their life, cells must maintain homeostasis while facing constantly fluctuating demands on their different organelles. A major mechanism for the homeostatic control of organelle function is the unfolded protein response (UPR), a signaling pathway that triggers a comprehensive remodeling of the endoplasmic reticulum (ER) and the biosynthetic pathway according to need. We discovered that activation of the UPR in yeast also induces a new branch of macroautophagy that selectively targets the ER. We term this process "ER-phagy", in analogy to pexophagy and mitophagy, the two other known forms of organelle-specific marcoautophagy. ER-phagy involves the generation of autophagosomes that selectively include ER membranes and whose delimiting double membranes also derive, at least in part, from the ER. This finding provides direct evidence that the ER can serve as a membrane source for autophagosome formation and indicates that ER-phagy entails engulfment of the ER by itself. ER-phagy could remove damaged or redundant parts of the ER and thus represent an important degradative functionality of the UPR that helps to afford homeostatic control.     

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.Subject terms: Cell biology, Cancer     

Characterization of constitutive ER-phagy of excess membrane proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Endosomal sorting complexes required for transport-0 (ESCRT-0) are essential for fungal development,pathogenicity, autophagy and ER-phagy in Magnaporthe oryzae

Magnaporthe oryzae is an important plant pathogen that causes rice blast. Hse1 and Vps27 are components of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway and biogenesis. To date, the biological functions of ESCRT-0 in M. oryzae have not been determined. In this study, we identified and characterized Hse1 and Vps27 in M. oryzae. Disruption of MoHse1 and MoVps27 caused pleiotropic defects in growth, conidiation, sexual development and pathogenicity, thereby resulting in loss of virulence in rice and barley leaves. Disruption of MoHse1 and MoVps27 triggered increased lipidation of MoAtg8 and degradation of GFP-MoAtg8, indicating that ESCRT-0 is involved in the regulation of autophagy. ESCRT-0 was determined to interact with coat protein complex II (COPII), a regulator functioning in homeostasis of the endoplasmic reticulum (ER homeostasis), and disruption of MoHse1 and MoVps27 also blocked activation of the unfolded protein response (UPR) and autophagy of the endoplasmic reticulum (ER-phagy). Overall, our results indicate that ESCRT-0 plays critical roles in regulating fungal development, virulence, autophagy and ER-phagy in M. oryzae.     

Regulation of ER-phagy by a Ypt/Rab GTPase module

Accumulation of misfolded proteins on intracellular membranes has been implicated in neurodegenerative diseases. One cellular pathway that clears such aggregates is endoplasmic reticulum autophagy (ER-phagy), a selective autophagy pathway that delivers excess ER to the lysosome for degradation. Not much is known about the regulation of ER-phagy. The conserved Ypt/Rab GTPases regulate all membrane trafficking events in eukaryotic cells. We recently showed that a Ypt module, consisting of Ypt1 and autophagy-specific upstream activator and downstream effector, regulates the onset of selective autophagy in yeast. Here we show that this module acts at the ER. Autophagy-specific mutations in its components cause accumulation of excess membrane proteins on aberrant ER structures and induction of ER stress. This accumulation is due to a block in transport of these membranes to the lysosome, where they are normally cleared. These findings establish a role for an autophagy-specific Ypt1 module in the regulation of ER-phagy. Moreover, because Ypt1 is a known key regulator of ER-to-Golgi transport, these findings establish a second role for Ypt1 at the ER. We therefore propose that individual Ypt/Rabs, in the context of distinct modules, can coordinate alternative trafficking steps from one cellular compartment to different destinations.     

Autophagy: an ER Protein Quality Control Process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome–dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both a1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.

Addendum to:

Characterization of an ERAD Gene as VPS30/ATG6 Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble A1PiZ and Another for Aggregates of A1PiZ

K.B. Kruse, J.L. Brodsky and A.A. McCracken

Mol Biol Cell 2005; In press.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/Co2+-dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.     

Endoplasmic reticulum stress response in yeast and humans

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.     

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.KEY WORDS: Unfolded protein response, Steatosis, Zebrafish, Tunicamycin, Thapsigargin, ER stress, Fatty liver disease