CALCOCO1 acts with VAMP-associated proteins to mediate ER-phagy.

The endoplasmic reticulum (ER) plays important roles in protein synthesis and folding, and calcium storage. The volume of the ER and expression of its resident proteins are increased in response to nutrient stress. ER-phagy, a selective form of autophagy, is involved in the degradation of the excess components of the ER to restore homeostasis. Six ER-resident proteins have been identified as ER-phagy receptors so far. In this study, we have identified CALCOCO1 as a novel ER-phagy receptor for the degradation of the tubular ER in response to proteotoxic and nutrient stress. CALCOCO1 is a homomeric protein that binds directly to ATG8 proteins via LIR- and UDS-interacting region (UIR) motifs acting co-dependently. CALCOCO1-mediated ER-phagy requires interaction with VAMP-associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT-like motif. Depletion of CALCOCO1 causes expansion of the ER and inefficient basal autophagy flux. Unlike the other ER-phagy receptors, CALCOCO1 is peripherally associated with the ER. Therefore, we define CALCOCO1 as a soluble ER-phagy receptor.

NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1.

The tripartite motif (TRIM) proteins constitute a family of ubiquitin E3 ligases involved in a multitude of cellular processes, including protein homeostasis and autophagy. TRIM32 is characterized by six protein-protein interaction domains termed NHL, various point mutations in which are associated with limb-girdle-muscular dystrophy 2H (LGMD2H). Here, we show that TRIM32 is an autophagy substrate. Lysosomal degradation of TRIM32 was dependent on ATG7 and blocked by knockout of the five autophagy receptors p62 (also known as SQSTM1), NBR1, NDP52 (also known as CALCOCO2), TAX1BP1 and OPTN, pointing towards degradation by selective autophagy. p62 directed TRIM32 to lysosomal degradation, while TRIM32 mono-ubiquitylated p62 on lysine residues involved in regulation of p62 activity. Loss of TRIM32 impaired p62 sequestration, while reintroduction of TRIM32 facilitated p62 dot formation and its autophagic degradation. A TRIM32LGMD2H disease mutant was unable to undergo autophagic degradation and to mono-ubiquitylate p62, and its reintroduction into the TRIM32-knockout cells did not affect p62 dot formation. In light of the important roles of autophagy and p62 in muscle cell proteostasis, our results point towards impaired TRIM32-mediated regulation of p62 activity as a pathological mechanisms in LGMD2H.

TRIM50 regulates Beclin 1 proautophagic activity.

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.

FKBP8 recruits LC3A to mediate Parkin-independent mitophagy.

Mitophagy, the selective removal of damaged or excess mitochondria by autophagy, is an important process in cellular homeostasis. The outer mitochondrial membrane (OMM) proteins NIX, BNIP3, FUNDC1, and Bcl2-L13 recruit ATG8 proteins (LC3/GABARAP) to mitochondria during mitophagy. FKBP8 (also known as FKBP38), a unique member of the FK506-binding protein (FKBP) family, is similarly anchored in the OMM and acts as a multifunctional adaptor with anti-apoptotic activity. In a yeast two-hybrid screen, we identified FKBP8 as an ATG8-interacting protein. Here, we map an N-terminal LC3-interacting region (LIR) motif in FKBP8 that binds strongly to LC3A both in vitro and in vivo FKBP8 efficiently recruits lipidated LC3A to damaged mitochondria in a LIR-dependent manner. The mitophagy receptors BNIP3 and NIX in contrast are unable to mediate an efficient recruitment of LC3A even after mitochondrial damage. Co-expression of FKBP8 with LC3A profoundly induces Parkin-independent mitophagy. Strikingly, even when acting as a mitophagy receptor, FKBP8 avoids degradation by escaping from mitochondria. In summary, this study identifies novel roles for FKBP8 and LC3A, which act together to induce mitophagy.

Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress.

BACKGROUND: The transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression. METHODS: The CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents. RESULTS: The knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells. CONCLUSIONS: The U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB.

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element in the p62 promoter. Second, p62 accumulation, occurring when autophagy is impaired, leads to increased p62 binding to the NRF2 inhibitor KEAP1, resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. Here, we show that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via an LC3-interacting region motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional antioxidant response element that is directly bound by the NRF2 orthologue, CncC, which can induce ref(2)P expression along with the oxidative stress-associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region motif; nor does ectopically expressed Ref(2)P or autophagy deficiency activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress-sensing NRF2/CncC transcription factors and autophagy and suggest that NRF2/CncC may regulate autophagic activity in other organisms too.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP.

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551-1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551-1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551-1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith-Magenis syndrome and Potocki-Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

TRIM27 is an autophagy substrate facilitating mitochondria clustering and mitophagy via phosphorylated TBK1.

Tripartite motif-containing protein 27 (TRIM27/also called RFP) is a multifunctional ubiquitin E3 ligase involved in numerous cellular functions, such as proliferation, apoptosis, regulation of the NF-kB pathway, endosomal recycling and the innate immune response. TRIM27 interacts directly with TANK-binding kinase 1 (TBK1) and regulates its stability. TBK1 in complex with autophagy receptors is recruited to ubiquitin chains assembled on the mitochondrial outer membrane promoting mitophagy. Here, we identify TRIM27 as an autophagy substrate, depending on ATG7, ATG9 and autophagy receptors for its lysosomal degradation. We show that TRIM27 forms ubiquitylated cytoplasmic bodies that co-localize with autophagy receptors. Surprisingly, we observed that induced expression of EGFP-TRIM27 in HEK293 FlpIn TRIM27 knockout cells mediates mitochondrial clustering. TRIM27 interacts with autophagy receptor SQSTM1/p62, and the TRIM27-mediated mitochondrial clustering is facilitated by SQSTM/p62. We show that phosphorylated TBK1 is recruited to the clustered mitochondria. Moreover, induced mitophagy activity is reduced in HEK293 FlpIn TRIM27 knockout cells, while re-introduction of EGFP-TRIM27 completely restores the mitophagy activity. Inhibition of TBK1 reduces mitophagy in HEK293 FlpIn cells and in the reconstituted EGFP-TRIM27-expressing cells, but not in HEK293 FlpIn TRIM27 knockout cells. Altogether, these data reveal novel roles for TRIM27 in mitophagy, facilitating mitochondrial clustering via SQSTM1/p62 and mitophagy via stabilization of phosphorylated TBK1 on mitochondria.

Transforming growth factor-ß-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs.

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.

p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription.

The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, is up-regulated by various stressors. Induction of the p62 gene by oxidative stress is mediated by NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to the activation of NRF2, but hitherto the mechanisms involved were not known. Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter that is responsible for its induction by oxidative stress via NRF2. Chromatin immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to this cis-element in vivo and in vitro. Also, p62 docks directly onto the Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif designated the KEAP1 interacting region (KIR), thereby blocking binding between KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription factor. The KIR motif in p62 is located immediately C-terminal to the LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell. The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but because p62 is polymeric the interaction between KEAP1 and p62 leads to accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes in response to oxidative stress through creating a positive feedback loop.

Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations.

The transcription factor Pax6 is crucial for the embryogenesis of multiple organs, including the eyes, parts of the brain and the pancreas. Mutations in one allele of PAX6 lead to eye diseases including Peter's anomaly and aniridia. Here, we use fluorescence recovery after photobleaching to show that Pax6 and also other Pax family proteins display a strikingly low nuclear mobility compared to other transcriptional regulators. For Pax6, the slow mobility is largely due to the presence of two DNA-binding domains, but protein-protein interactions also contribute. Consistently, the subnuclear localization of Pax6 suggests that it interacts preferentially with chromatin-rich territories. Some aniridia-causing missense mutations in Pax6 have impaired DNA-binding affinity. Interestingly, when these mutants were analyzed by FRAP, they displayed a pronounced increased mobility compared to wild-type Pax6. Hence, our results support the conclusion that disease mutations result in proteins with impaired function because of altered DNA- and protein-interaction capabilities.

Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence.

TRIM32 is an E3 ligase implicated in diverse biological pathways and pathologies such as muscular dystrophy and cancer. TRIM32 are expressed both as full-length proteins, and as a truncated protein. The mechanisms for regulating these isoforms are poorly understood. Here we identify a PEST sequence in TRIM32 located in the unstructured region between the RING-BBox-CoiledCoil domains and the NHL repeats. The PEST sequence directs cleavage of TRIM32, generating a truncated protein similarly to the short isoform. We map three lysine residues that regulate PEST mediated cleavage and auto-ubiquitylation activity of TRIM32. Mimicking acetylation of lysine K247 completely inhibits TRIM32 cleavage, while the lysines K50 and K401 are implicated in auto-ubiquitylation activity. We show that the short isoform of TRIM32 is catalytic inactive, suggesting a dominant negative role. These findings uncover that TRIM32 is regulated by post-translational modifications of three lysine residues, and a conserved PEST sequence.

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity.

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.

The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription.

SPBP (Stromelysin-1 PDGF responsive element binding protein) is a ubiquitously expressed 220 kDa nuclear protein shown to enhance or repress the transcriptional activity of various transcription factors. A yeast two-hybrid screen, with the extended plant homeodomain (ePHD) of SPBP as bait, identified TopBP1 (topoisomerase II beta-binding protein 1) as a candidate interaction partner of SPBP. TopBP1 has eight BRCA1 carboxy-terminal (BRCT) domains and is involved in DNA replication, DNA damage responses and in the regulation of gene expression. The interaction between SPBP and TopBP1 was confirmed in vitro and in vivo, and was found to be mediated by the ePHD domain of SPBP and the BRCT6 domain of TopBP1. Both SPBP and TopBP1 enhanced the transcriptional activity of Ets1 on the c-myc P1P2- and matrix metalloproteinase-3 (MMP3) promoters. Together they displayed a more than additive effect. Both proteins were associated with these promoters. The involvement of TopBP1 was dependent on the serine 1159 phosphorylation site, known to be important for transcriptional activation. Depletion of endogenous SPBP by siRNA treatment reduced MMP3 secretion by 50% in phorbol ester-stimulated human fibroblasts. Taken together, our results show that TopBP1 and SPBP interact physically and functionally to co-operate as co-activators of Ets1.

The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG-repeat expansion in the 5' UTR of the FMR1 gene on the X-chromosome. Both elevated levels of the expanded FMR1 mRNA and aberrant expression of a polyglycine protein (FMRpolyG) from the CGG-repeat region are hypothesized to trigger the pathogenesis of FXTAS. While increased expression of FMRpolyG leads to higher toxicity in FXTAS models, the pathogenic effect of this protein has only been studied in the presence of CGG-containing mRNA. Here we present a model that allows measurement of the effect of FMRpolyG-expression without co-expression of the corresponding CGG mRNA hairpin. This allows direct comparison of the effect of the FMRpolyG protein per se, vs. that of the FMRpolyG protein together with the CGG mRNA hairpin. Our results show that expression of the FMRpolyG, in the absence of any CGG mRNA, is sufficient to cause reduced cell viability, lamin ring disruption and aggregate formation. Furthermore, we found FMRpolyG to be a long-lived protein degraded primarily by the ubiquitin-proteasome-system. Together, our data indicate that accumulation of FMRpolyG protein per se may play a major role in the development of FXTAS.

Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs.

Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation. Abbreviations: ATG: autophagy-related; BafA1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GFP: enhanced green fluorescent protein; KO: knockout; LDS: LIR docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62: sequestosome 1; VPS: Vacuolar protein sorting; ULK: unc-51 like autophagy activating kinase.

The recent transfer of a homing endonuclease gene.

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.