GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP‐LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP‐LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP‐Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin‐positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP‐LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP‐LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP‐LC3, a fusion protein widely used as a marker for autophagic vesicles and pre‐autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular cloning and characterization of rat LC3A and LC3B--two novel markers of autophagosome

Rat microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8, an essential component of autophagy. Following synthesis, the C-terminus of rat LC3 is cleaved by a cysteine protease-Atg4, to produce LC3-I, which is located in cytosolic fraction. LC3-I can be converted to LC3-II through the processing by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). LC3-II is modified by phosphatidylethanolamine on C-terminus and binds tightly to autophagosomal membrane. Here we reported the cloning of two novel variants of rat LC3, named LC3A and LC3B, respectively, and LC3B is an alternative splicing variant of LC3. LC3A, LC3B, and LC3 showed different expression patterns in rat tissues, suggesting a functional divergence among these proteins. When LC3A and LC3B were overexpressed, both exhibited two forms (18 and 16 kDa, representing types of I and II, separately), which might be due to post-translational modification including the characteristic C-terminal cleavage at these two proteins as similar to that found in rat LC3 and yeast Atg8. Subcellular localization demonstrated that both LC3A and LC3B are colocalized with LC3 and associated with the autophagic membranes. Mutation analysis further revealed that the conserved Gly120 residues of LC3A and LC3B are essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that LC3A and LC3B could also be used as two novel autophagosomal markers.     

Processing of autophagic protein LC3 by the 20S proteasome

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.     

Binding preference of p62 towards LC3-ll during dopaminergic neurotoxin-induced impairment of autophagic flux

Accumulating evidence has revealed that autophagy may be beneficial for treatment of neurodegenerative diseases through removal of abnormal protein aggregates. However, the critical autophagic events during neurodegeneration remain to be elucidated. Here, we investigated whether prototypic autophagic events occur in the MN9D dopaminergic neuronal cell line upon exposure to N-methyl-4-phenylpyridinium (MPP (+) ), a well-known dopaminergic neurotoxin. MPP (+) treatment induced both morphological and biochemical characteristics of autophagy, such as accumulation of autophagic vacuoles and LC3-II form and decreased p62 levels. Further investigation revealed that these phenomena were largely the consequences of blocked autophagic flux. Following MPP (+) treatment, levels of LC3-II formed and p62 dramatically increased in the Triton X-100-insoluble fraction. Levels of ubiquitinated proteins also increased in this fraction. Further colocalization analyses revealed that the punctated spots positive for both p62 and LC3 were more intense following MPP (+) treatment, suggesting drug-induced enrichment of these two proteins in the insoluble fraction. Intriguingly, reciprocal immunoprecipitation analysis revealed that p62 mainly precipitated with LC3-II form following MPP (+) treatment. Transient transfection of the mutant form of Atg4B, Atg4B (C74A) , which inhibits LC3 processing, dramatically decreased binding between p62 and LC3-II form. Taken together, our results indicate that p62 can be efficiently localized to autophagic compartments via preferential binding with LC3-II form. This colocalization may assist in removal of detergent-insoluble forms of damaged cellular proteins during dopaminergic neurotoxin-induced impairment of autophagic flux.     

Protein kinase C inhibits autophagy and phosphorylates LC3

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Mycobacterium marinum induces a marked LC3 recruitment to its containing phagosome that depends on a functional ESX-1 secretion system

Autophagy has been implicated as part of the innate immune system against different intracellular microorganisms. Mycobacterium marinum is the causative agent of the fish-tank granuloma and has been widely used as an alternative model to study pathogenic mycobacteria. In this report, we show an active interaction of M. marinum with the autophagic protein LC3, an event that requires pathogen viability and bacterial protein synthesis. Interestingly, M. marinum lacking the region of difference 1 (RD1) is unable to recruit LC3, indicating that a functional ESX-1 secretion system is an absolute requirement for this process. In addition, phagocytosis of the bacteria is also a condition for the LC3 rearrangement induced by M. marinum. We present evidence that this pathogen resides temporarily in a LC3-decorated compartment with late endocytic features but mostly devoid of lysosomal enzymes or degradative properties. In addition our results indicate that autophagy induction by rapamycin treatment leads to maturation of the M. marinum-containing compartment.     

Autophagosome Proteins LC3A,LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.     

"Autophagic flux" in normal mouse tissues: Focus on endogenous LC3A processing

Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best studied autophagic marker is the LC3B and it is believed that only the amount of the LC3B-II correlates with the amount of the autophagic membranes. Whether the LC3A processing, aside to LC3B, is a valuable endogenous 'autophagic flux' marker is far less clear. The specificity of rabbit polyclonal antibodies to the LC3A and the LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure 'autophagic flux' in mouse liver, lung, kidney and heart we used: i. a lysosomotropic reagent chloroquine, which inhibit the intra-lysosomal acidification or their fusion with autophagosome, ii. nutrient starvation as an autophagic stimulus and iii. ionizing radiation, which is known to destabilize lysosomes. According to the immunoblotting work the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein didn't follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in mouse model. Fractionated samples (soluble vs. membrane fractions) should be used in immunobloting to allow discrimination between the LC3-I soluble and the LC3-II membrane protein and kinetics. Further, when dealing with in vivo models it is necessary to check the specificity of the antibodies used against the LC3A and LC3B proteins as their expression and responsiveness is not overlapping.     

Using LC3 to monitor autophagy flux in the retinal pigment epithelium

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation.^1,2 Autophagy begins with the formation of membranes arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. ^1,2,7 However, few studies of autophagy in retinal disease have been reported.

Recent studies show that autophagy and changes in lysosomal activity are associated with both retinal aging and age-related macular degeneration (AMD).^3,4 This article describes methods which employ the target protein LC3 to monitor autophagic flux in retinal pigment epithelial cells. During autophagy, the cytosolic form of LC3 (LC3-I) is processed and recruited to the phagophore where it undergoes site specific proteolysis and lipidation near the C terminus to form LC3-II.⁵ Monitoring the formation of cellular autophagosome puncta containing LC3 and measuring the ratio of LC3-II to LC3-I provides the ability to monitor autophagy flux in the retina.     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

Autophagy in normal tissues of camel (Camelus dromedarius) with focus on immunoexpression of LC3 and LC3B

Autophagy is a highly regulated intracellular pathway for degradation and recycling of cytoplasmic protein aggregates and entire organelles. The autophagic pathway is stimulated by nutrient starvation, which prompted us to study the desert camel. Various organs of the camel undergo ecological and physiological stress due to food and water deprivation, dehydration and long exposure to solar radiation. We investigated the immunohistochemical expression of specific biomarkers of autophagy under normal conditions as a baseline for later work on stressed individuals. The autophagy-specific biomarkers, microtubule-associated protein1 light chain 3 (LC3), and its cleaved variant, LC3B, were strongly expressed in the cytosol of all tissues examined. The cytosolic immunoreactivity of LC3 was relatively weak, diffuse and vacuolar, while that of LC3B was stronger, punctate and at lower levels. LC3 appears to be associated with the autophagosomal membranes, either free or lysosome-bounded. LC3B is specific for the autophagosome-lysosome complexes and their degraded, granular contents. Autophagy was strongly expressed in CNS neurons and intestinal neural elements, which suggests a protective function for the nervous system. Autophagic markers also were seen in deformed immune-competent cells with fragmented nuclei in lymph nodes, spleen and gut-associated lymphoid tissue (GALT), which suggests a “suicidal” activity of eliminating unneeded cells. Autophagy, as measured by LC3 and LC3B expression, may participate in a general regulatory mechanism in tissues of the desert camel.     

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.     

How to Interpret LC3 Immunoblotting

Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.     

How to interpret LC3 immunoblotting

Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.     

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.     

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.     

Sequence-specific <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the autophagy-related protein LC3C

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific ¹H, ¹⁵N, and ¹³C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy.     

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.