板倉 英祐

The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding and post-translational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, i.e., selective autophagy of the ER, is a mechanism that maintains or re-establishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate, and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo interaction regions (CIRs); these directly interact with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 directly interact with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy.

Abstract Autophagy plays important role in the intracellular protein quality control system by degrading abnormal organelles and proteins, including large protein complexes such as ribosomes. The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC), also called chaperonin-containing TCP1 (CCT), is a 1-MDa hetero-oligomer complex comprising 16 subunits that facilitates the folding of ~10% of the cellular proteome that contains actin. However, the quality control mechanism of TRiC remains unclear. To monitor the autophagic degradation of TRiC, we generated TCP1α-RFP-GFP knock-in HeLa cells using a CRISPR/Cas9-knock-in system with an RFP-GFP donor vector. We analyzed the autophagic degradation of TRiC under several stress conditions and found that treatment with actin (de)polymerization inhibitors increased the lysosomal degradation of TRiC, which was localized in lysosomes and suppressed by deficiency of autophagy-related genes. Furthermore, we found that treatment with actin (de)polymerization inhibitors increased the association between TRiC and unfolded actin, suggesting that TRiC was inactivated. Moreover, unfolded actin mutants were degraded by autophagy. Taken together, our results indicate that autophagy eliminates inactivated TRiC, serving as a quality control system.

Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).

Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enable the labeling of stressed mitochondria. Mito-Pain uses PTEN-induced putative kinase 1 (PINK1) stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provides insights into mitochondrial quality control systems.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

The accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor. Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid β peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.

Autophagy is an intracellular process that regulates the degradation of cytosolic proteins and organelles. Dying cells often accumulate autophagosomes. However, the mechanisms by which necroptotic stimulation induces autophagosomes are not defined. Here, we demonstrate that the activation of necroptosis with TNF-α plus the cell-permeable pan-caspase inhibitor Z-VAD induces LC3-II and LC3 puncta, markers of autophagosomes, via the receptor-interacting protein kinase 3 (RIPK3) in intestinal epithelial cells. Surprisingly, necroptotic stimulation reduces autophagic activity, as evidenced by enlarged puncta of the autophagic substrate SQSTM1/p62 and its increased colocalization with LC3. However, necroptotic stimulation does not induce the lysosomal-associated membrane protein 1 (LAMP1) nor syntaxin 17, which mediates autophagosome-lysosome fusion, to colocalize with LC3. These data indicate that necroptosis attenuates autophagic flux before the lysosome fusion step. Our findings may provide insights into human diseases involving necroptosis.

Lysosomes are largely responsible for significant degradation of intracellular and extracellular proteins via the secretory pathway, autophagy, and endocytosis. Therefore, dysregulation of lysosomal homeostasis influences diverse cellular functions. However, a straightforward and quantitative method to measure the integrity of the lysosomal pathway has not been developed. Here, we report the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe that enables simple quantification of lysosomal integrity by lysosomal green and cytosolic red fluorescent proteins using a flow cytometer. In cultured cells, the lysosomal-METRIQ probe detected not only suppression of the lysosomal pathway but also upregulation of lysosomal activity such as lysosomal biogenesis. To identify factors involved in lysosomal homeostasis, we carried out compound screening and found that the cyclin-dependent kinase (CDK) inhibitors kenpaullone and purvalanol A induce synthesis of cathepsin D and an increase in the number of lysosomes. Subsequent studies revealed that CDK5 maintains lysosomal homeostasis independently of cell cycle arrest. Our results suggest that the lysosomal-METRIQ probe is an effective and efficient tool for measuring lysosomal activity in mammalian cells.

The telomere protects the ends of eukaryotic linear chromosomes, and its shortening or erosion is recognized as DNA damage, leading to loss of proliferation activity and, thus, cellular senescence at the population level. Here, using a GFP-based DNA damage checkpoint marker suited for single-cell observation of Saccharomyces cerevisiae cells, we correlated the checkpoint status of telomere-shortened cells with their behavior. We show that some cells possessing short telomeres retain proliferation capacity even after the DNA damage checkpoint is activated. At the presenescent stage, the activation of the checkpoint causes cell cycle delay, but does not induce permanent cell cycle arrest, eventually leading to the expansion of cell size that is characteristic of cellular senescence. Moreover, the proliferation capacity of checkpoint-activated cells is not dependent on homologous recombination or the checkpoint adaptation pathway. The retention of proliferation capacity is specific to the telomere-derived DNA damage response, suggesting that damaged telomeres differ functionally from other types of DNA damage. Our data establish the role of the presenescent stage in telomere shortening-induced senescence, which proceeds gradually and is associated with a variety of changes, including altered cell morphology and metabolism.

Ras-activated ERK pathway (Raf-MEK-ERK phosphorylation cascade) regulates a variety of cellular responses including cell proliferation, differentiation, survival, and apoptosis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner. Here we show that DA-Raf plays essential roles in skeletal myocyte differentiation including myoblast fusion and in apoptosis, which are suppressed by the Ras-ERK pathway. Expression of DA-Raf was highly induced in C2C12 skeletal myocytes in a low serum concentration of differentiation condition and in NIH3T3 fibroblasts under a serum starvation apoptosis-inducing condition. Stable knockdown of DA-Raf resulted in suppression of muscle-specific gene expression, myoblast fusion, and apoptosis. In contrast, exogenous overexpression of DA-Raf prominently caused apoptosis. DA-Raf induces apoptosis by preventing ERK-RSK-mediated inhibitory phosphorylation of Bad. Although it has been reported that apoptosis triggers myoblast fusion, DA-Raf-induced apoptosis was not involved in myoblast fusion in C2C12 cells. These results imply that suppression of the Ras-ERK pathway by DA-Raf is essential for both myocyte differentiation including myoblast fusion and apoptosis but that apoptosis is not a prerequisite for myoblast fusion.

Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.

Target of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, Saccharomyces cerevisiae, TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.

This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.

Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy.

Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy.

Interleukin-17 (IL-17) is a major pro-inflammatory cytokine: it mediates responses to pathogens or tissue damage, and drives autoimmune diseases. Little is known about its role in the nervous system. Here we show that IL-17 has neuromodulator-like properties in Caenorhabditis elegans. IL-17 can act directly on neurons to alter their response properties and contribution to behaviour. Using unbiased genetic screens, we delineate an IL-17 signalling pathway and show that it acts in the RMG hub interneurons. Disrupting IL-17 signalling reduces RMG responsiveness to input from oxygen sensors, and renders sustained escape from 21% oxygen transient and contingent on additional stimuli. Over-activating IL-17 receptors abnormally heightens responses to 21% oxygen in RMG neurons and whole animals. IL-17 deficiency can be bypassed by optogenetic stimulation of RMG. Inducing IL-17 expression in adults can rescue mutant defects within 6 h. These findings reveal a non-immunological role of IL-17 modulating circuit function and behaviour.

Autophagy is a cytoplasmic degradation system that is important for starvation adaptation and cellular quality control. Previously, we reported that Atg5-null mice are neonatal lethal; however, the exact cause of their death remains unknown. Here, we show that restoration of ATG5 in the brain is sufficient to rescue Atg5-null mice from neonatal lethality. This suggests that neuronal dysfunction, including suckling failure, is the primary cause of the death of Atg5-null neonates, which would further be accelerated by nutrient insufficiency due to a systemic failure in autophagy. The rescued Atg5-null mouse model, as a resource, allows us to investigate the physiological roles of autophagy in the whole body after the neonatal period. These rescued mice demonstrate previously unappreciated abnormalities such as hypogonadism and iron-deficiency anemia. These observations provide new insights into the physiological roles of the autophagy factor ATG5.

We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions.

Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts (MEFs) and HeLa cells deficient in various ATG proteins reveals that the emergence of the isolation membrane (phagophore) requires FIP200 (also known as RB1CC1), ATG9A and phosphatidylinositol (PtdIns) 3-kinase activity. By contrast, small premature isolation-membrane-like and autophagosome-like structures were generated in cells lacking VMP1 and both ATG2A and ATG2B, respectively. The isolation membranes could elongate in cells lacking ATG5, but did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62 (also known as SQSTM1) in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the site of autophagosome formation.

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.

Proteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. Here, we report that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.

Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome-lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome-lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar protein sorting 33A (VPS33A) and VPS16, which are components of the homotypic fusion and protein sorting (HOPS)-tethering complex. We further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and microtubule-associated protein light chain (LC3)-positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, ultraviolet irradiation resistance-associated gene (UVRAG), a known HOPS-interacting protein, did not interact with the STX17-HOPS complex and may not be directly involved in autophagosome-lysosome fusion. Collectively these results suggest that, in addition to its well-established function in the endocytic pathway, HOPS promotes autophagosome-lysosome fusion through interaction with STX17.

SQSTM1/p62 (sequestosome 1) is a multifunctional signaling molecule, involved in a variety of cellular pathways. SQSTM1 is one of the best-known autophagic substrates, and is therefore widely used as an indicator of autophagic degradation. Here we report that the expression level of SQSTM1 can be restored during prolonged starvation. Upon starvation, SQSTM1 is initially degraded by autophagy. However, SQSTM1 is restored back to basal levels during prolonged starvation in mouse embryonic fibroblasts and HepG2 cells, but not in HeLa and HEK293 cells. Restoration of SQSTM1 depends on its transcriptional upregulation, which is triggered by amino acid starvation. Furthermore, amino acids derived from the autophagy-lysosome pathway are used for de novo synthesis of SQSTM1 under starvation conditions. The restoration of SQSTM1 is independent of reactivation of MTORC1 (mechanistic target of rapamycin complex 1). These results suggest that the expression level of SQSTM1 in starved cells is determined by at least 3 factors: autophagic degradation, transcriptional upregulation, and availability of lysosomal-derived amino acids. The results of this study also indicate that the expression level of SQSTM1 does not always inversely correlate with autophagic activity.

Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex's role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis.

Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.

The phagophore (also called isolation membrane) elongates and encloses a portion of cytoplasm, resulting in formation of the autophagosome. After completion of autophagosome formation, the outer autophagosomal membrane becomes ready to fuse with the lysosome for degradation of enclosed cytoplasmic materials. However, the molecular mechanism for how the fusion of completed autophagosomes with the lysosome is regulated has not been fully understood. We discovered syntaxin 17 (STX17) as an autophagosomal soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE). STX17 has a hairpin-type structure mediated by two transmembrane domains, each containing glycine zipper motifs. This unique transmembrane structure contributes to its specific localization to completed autophagosomes but not to phagophores. STX17 interacts with SNAP29 and the lysosomal SNARE VAMP8, and all of these proteins are required for autophagosome-lysosome fusion. The late recruitment of STX17 to completed autophagosomes could prevent premature fusion of the lysosome with unclosed phagophores. © 2013 Landes Bioscience.

The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome.

Pituitary folliculostellate (FS) cells are characterized by producing S100B protein, as do brain astrocytes. FS cells have some functions in the pituitary gland, i.e. scavenger functions, sustentacular cell activity through cytokines, and intercellular communication through gap junctions. However, the biological significances of FS cells, especially their differentiation capacities in the anterior pituitary gland, are still under discussion. To understand FS cells with new approaches, we generated a transgenic rat expressing GFP under S100b gene promoter, which regulates tissue-specific expression of S100b gene. Using the transgenic rat, we succeeded in inducing skeletal muscle cells from FS cells by culturing it in serum-free medium containing B-27 supplement, thyroid hormone (tri-iodothyronine), epidermal growth factor, and basic fibroblast growth factor. In this study, we also succeeded in inducing skeletal muscle cells from primary cultured astrocytes and astrocyte cell line, C6 cells. Hence, we concluded that pituitary FS cells have wide differentiation potential and have similar characteristics to astrocytes, which not only support cell activity but also support differentiation capacity. Journal of Endocrinology (2012) 213, 231-237

Mitochondria can be degraded by autophagy in a process termed mitophagy. The Parkinson-disease-associated ubiquitin ligase Parkin can trigger mitophagy of depolarized mitochondria. However, it remains to be determined how the autophagy machinery is involved in this specific type of autophagy. It has been speculated that adaptor proteins such as p62 might mediate the interaction between the autophagosomal LC3 family of proteins and ubiquitylated proteins on mitochondria. Here, we describe our systematic analysis of the recruitment of Atg proteins in Parkin-dependent mitophagy. Structures containing upstream Atg proteins, including ULK1, Atg14, DFCP1, WIPI-1 and Atg16L1, can associate with depolarized mitochondria even in the absence of membrane-bound LC3. Atg9A structures are also recruited to these damaged mitochondria as well as to the autophagosome formation site during starvation-induced canonical autophagy. In the initial steps of Parkin-mediated mitophagy, the structures containing the ULK1 complex and Atg9A are independently recruited to depolarized mitochondria and both are required for further recruitment of downstream Atg proteins except LC3. Autophagosomal LC3 is important for efficient incorporation of damaged mitochondria into the autophagosome at a later stage. These findings suggest a process whereby the isolation membrane is generated de novo on damaged mitochondria as opposed to one where a preformed isolation membrane recognizes mitochondria.

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.

Autophagy is an intracellular degradation process, through which cytosolic materials are delivered to the lysosome. Despite recent identification of many autophagy-related genes, how autophagosomes are generated remains unclear. Here, we examined the hierarchical relationships among mammalian Atg proteins. Under starvation conditions, ULK1, Atg14, WIPI-1, LC3 and Atg16L1 target to the same compartment, whereas DFCP1 localizes adjacently to these Atg proteins. In terms of puncta formation, the protein complex including ULK1 and FIP200 is the most upstream unit and is required for puncta formation of the Atg14-containing PI3-kinase complex. Puncta formation of both DFCP1 and WIPI-1 requires FIP200 and Atg14. The Atg12-Atg5-Atg16L1 complex and LC3 are downstream units among these factors. The punctate structures containing upstream Atg proteins such as ULK1 and Atg14 tightly associate with the ER, where the ER protein vacuole membrane protein 1 (VMP1) also transiently localizes. These structures are formed even when cells are treated with wortmannin to suppress autophagosome formation. These hierarchical analyses suggest that ULK1, Atg14 and VMP1 localize to the ER-associated autophagosome formation sites in a PI3-kinase activity-independent manner.

Vps34, a Class III phosphatidylinositol 3-kinase (PI3-kinase), produces phosphatidylinositol 3 phosphate (PI3P) and functions in various membrane traffic pathways including endocytosis, multivesicular body formation and autophagy. In mammalian cells, Vps34 forms a complex with Beclin 1, but it remains unclear how this Vps34 complex exerts its specific function on each membrane trafficking pathway. We recently identified mammalian Atg14, a new binding partner of the Vps34-Beclin 1 complex, using a computational approach. The Atg14 complex consists of Vps34, Beclin 1 and p150, but lacks UVRAG, which was previously reported to bind the Vps34-Beclin 1 complex. Atg14 localizes to isolation membrane/phagophore during starvation and is essential for autophagosome formation. In contrast, UVRAG primarily localizes to late endosomes. Since UVRAG shows homology with yeast Vps38, we speculate that it could be a mammalian Vps38 ortholog. These findings indicate that the Vps34-Beclin 1 complex has at least two distinct functions, which can be promoted by its binding partners 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.

Folliculo-stellate (FS) cells are known to act as sustentacular cells or scavenger cells in the anterior lobe. However, the precise function and origin of FS cells are still under discussion. Like brain astrocytes, FS cells contain S-100 beta protein, and FS cells can be detected immunocytochemically using antibodies for S-100 beta protein after fixation; however, living FS cells can not be detected. The generation of transgenic rats expressing green fluorescent protein (GFP) under the control of S-100 beta protein gene promoter may allow the detection of living FS cells, which may be an excellent tool for the study of FS cells. With the aim of generation of transgenic rats, we analyzed the promoter activity of the S-100 beta gene and found that intron 1 is important for cell-specific expression of the S-100 beta gene. Therefore, we obtained a DNA construct containing GFP gene under a part of the S-100 promoter with intron 1. We transfected the construct into rat embryos and succeeded in generating transgenic rats. The transgenic rats expressed GFP in FS cells specifically in the anterior lobe. GFP is also expressed in other known S-100 beta-expressing cells, i.e. brain astrocytes, adipocytes, and chondrocytes. We believe that the newly generated transgenic rats will provide a new approach for the study of FS cells and other S-100 beta protein-producing cells.

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

PI-kinase-related protein kinase ATR forms a complex with ATRIP and plays pivotal roles in maintaining genome integrity. When DNA is damaged, the ATR-ATRIP complex is recruited to chromatin and is activated to transduce the checkpoint signal, but the precise kinase activation mechanism remains unknown. Here, we show that ATRIP is phosphorylated in an ATR-dependent manner after genotoxic stimuli. The serine 68 and 72 residues are important for the phosphorylation in vivo and are required exclusively for direct modification by ATR in vitro. Using phospho-specific antibody, we demonstrated that phosphorylated ATRIP accumulates at foci induced by DNA damage. Moreover, the loss of phosphorylation does not lead to detectable changes in the relocalization of ATRIP to nuclear foci nor in the activation of downstream effector proteins. Collectively, our results suggest that the ATR-mediated phosphorylation of ATRIP at Ser-68 and -72 is dispensable for the initial response to DNA damage. (C) 2004 Elsevier Inc. All rights reserved.