RESUMO
We talk to corresponding author Tomotake Kanki and co-first authors Tomoyuki Fukuda and Kentaro Furukawa about their paper "The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy" (this issue of Molecular Cell), their career paths, interests outside of their fields, and how they strike a work-life balance.
Assuntos
Proteínas Mitocondriais , Ubiquitina-Proteína Ligases , Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Mitofagia , Mitocôndrias/metabolismo , Dinâmica MitocondrialRESUMO
Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.
Assuntos
Autofagia , Mitofagia , Animais , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Lipídeos , Mamíferos/metabolismoRESUMO
Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.
Assuntos
Mitocôndrias Musculares/metabolismo , Mitofagia , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Elevação dos Membros Posteriores , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Inanição , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Meiose , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cromossomos/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Fosforilação , Transporte Proteico/genética , Proteínas Repressoras/metabolismoRESUMO
In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx-/- spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity.This article has an associated First Person interview with the first author of the paper.
Assuntos
Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espermatócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Pareamento Cromossômico , Instabilidade Genômica , Genômica , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas MutL/genética , Proteínas MutL/metabolismo , Recombinação Genética , Cromossomos Sexuais/genética , Cromossomos Sexuais/metabolismo , Espermatócitos/citologiaRESUMO
Cohesion between sister chromatids in mitotic and meiotic cells is promoted by a ring-shaped protein structure, the cohesin complex. The cohesin core complex is composed of four subunits, including two structural maintenance of chromosome (SMC) proteins, one α-kleisin protein, and one SA protein. Meiotic cells express both mitotic and meiosis-specific cohesin core subunits, generating cohesin complexes with different subunit composition and possibly separate meiotic functions. Here, we have analyzed the in vivo function of STAG3, a vertebrate meiosis-specific SA protein. Mice with a hypomorphic allele of Stag3, which display a severely reduced level of STAG3, are viable but infertile. We show that meiocytes in homozygous mutant Stag3 mice display chromosome axis compaction, aberrant synapsis, impaired recombination and developmental arrest. We find that the three different α-kleisins present in meiotic cells show different dosage-dependent requirements for STAG3 and that STAG3-REC8 cohesin complexes have a critical role in supporting meiotic chromosome structure and functions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona/metabolismo , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Meiose/genética , Proteínas Nucleares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centrômero/genética , Centrômero/ultraestrutura , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Cromossomos/ultraestrutura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Nucleares/genética , Ovário/ultraestrutura , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Testículo/ultraestrutura , CoesinasRESUMO
The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament-central-element junction structure that promotes synaptonemal complex formation and synapsis.
Assuntos
Complexo Sinaptonêmico/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/metabolismo , Proteínas de Ligação a DNA , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estágio Paquíteno , Ligação Proteica , Complexo Sinaptonêmico/ultraestruturaRESUMO
The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Complexo Sinaptonêmico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Espermatócitos/metabolismo , CoesinasRESUMO
During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3' UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5'cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs.
Assuntos
Fator de Ligação a CCAAT/genética , Protaminas/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Espermatogênese/genética , Animais , Fator de Ligação a CCAAT/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Polirribossomos , Protaminas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Testículo/metabolismoRESUMO
Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos/genética , Meiose/fisiologia , Mitose/fisiologia , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Células Germinativas , Humanos , Mamíferos , CamundongosRESUMO
Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.
Assuntos
Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Meiose/genética , Fosforilação , Recombinação Genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Masculino , Prófase Meiótica I/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Espermatócitos/metabolismo , Complexo SinaptonêmicoRESUMO
Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor.
Assuntos
Evolução Molecular , Genoma , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Fator de Transcrição Associado à Microftalmia/genética , Microftalmia/genética , Filogenia , Algoritmos , Sequência de Aminoácidos , Animais , Bactérias/genética , Bactérias/metabolismo , Linhagem Celular Tumoral , Mapeamento Cromossômico , Bases de Dados Genéticas , Fungos/genética , Fungos/metabolismo , Redes Reguladoras de Genes , Loci Gênicos , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/classificação , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Redes e Vias Metabólicas , Fator de Transcrição Associado à Microftalmia/classificação , Fator de Transcrição Associado à Microftalmia/metabolismo , Microftalmia/metabolismo , Microftalmia/patologia , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
TOR complex 1 (TORC1) is a multi-protein kinase complex that coordinates cellular growth with environmental cues. Recent studies have identified Pib2 as a critical activator of TORC1 in budding yeast. Here, we show that loss of Pib2 causes severe growth defects in fission yeast cells, particularly when basal TORC1 activity is diminished by hypomorphic mutations in tor2, the gene encoding the catalytic subunit of TORC1. Consistently, TORC1 activity is significantly compromised in the tor2 hypomorphic mutants lacking Pib2. Moreover, as in budding yeast, fission yeast Pib2 localizes to vacuolar membranes via its FYVE domain, with its tail motif indispensable for TORC1 activation. These results strongly suggest that Pib2-mediated positive regulation of TORC1 is evolutionarily conserved between the two yeast species.
RESUMO
Mitochondria undergo fission and fusion, and their coordinated balance is crucial for maintaining mitochondrial homeostasis. In yeast, the dynamin-related protein Dnm1 is a mitochondrial fission factor acting from outside the mitochondria. We recently reported the mitochondrial intermembrane space protein Atg44/mitofissin/Mdi1/Mco8 as a novel fission factor, but the relationship between Atg44 and Dnm1 remains elusive. Here, we show that Atg44 is required to complete Dnm1-mediated mitochondrial fission under homeostatic conditions. Atg44-deficient cells often exhibit enlarged mitochondria with accumulated Dnm1 and rosary-like mitochondria with Dnm1 foci at constriction sites. These mitochondrial constriction sites retain the continuity of both the outer and inner membranes within an extremely confined space, indicating that Dnm1 is unable to complete mitochondrial fission without Atg44. Moreover, accumulated Atg44 proteins are observed at mitochondrial constriction sites. These findings suggest that Atg44 and Dnm1 cooperatively execute mitochondrial fission from inside and outside the mitochondria, respectively.Abbreviation: ATG: autophagy related; CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; ERMES: endoplasmic reticulum-mitochondria encounter structure; GA: glutaraldehyde; GFP: green fluorescent protein; GTP: guanosine triphosphate: IMM: inner mitochondrial membrane; IMS: intermembrane space; OMM: outer mitochondrial membrane; PB: phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RFP: red fluorescent protein; WT: wild type.
RESUMO
Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.
Assuntos
Ferroptose , Proteínas de Membrana , Mitocôndrias , Mitofagia , Proteínas Proto-Oncogênicas , Espécies Reativas de Oxigênio , Humanos , Regulação para Baixo , Células HeLa , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Most autophagy-related genes, or ATG genes, have been identified in studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy, and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway, and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research.
RESUMO
ABBREVIATIONS: Atg: autophagy related; IMM: inner mitochondrial membrane; IMS: intermembrane space; PAS: phagophore assembly site; SAR: selective autophagy receptor.
Assuntos
Autofagia , Mitofagia , Dinâmica Mitocondrial , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
The endoplasmic reticulum (ER) undergoes selective autophagy called reticulophagy or ER-phagy. Multiple reticulon- and receptor expression enhancing protein (REEP)-like ER-shaping proteins, including budding yeast Atg40, serve as reticulophagy receptors that stabilize the phagophore on the ER by interacting with phagophore-conjugated Atg8. Additionally, they facilitate phagophore engulfment of the ER by remodeling ER morphology. We reveal that Hva22, a REEP family protein in fission yeast, promotes reticulophagy without Atg8-binding capacity. The role of Hva22 in reticulophagy can be replaced by expressing Atg40 independently of its Atg8-binding ability. Conversely, adding an Atg8-binding sequence to Hva22 enables it to substitute for Atg40 in budding yeast. Thus, the phagophore-stabilizing and ER-shaping activities, both of which Atg40 solely contains, are divided between two separate factors, receptors and Hva22, respectively, in fission yeast.Abbreviations: AIM: Atg8-family interacting motif; Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum GFP: green fluorescent protein; NAA: 1-naphthaleneacetic acid; REEP: receptor expression enhancing protein; RFP: red fluorescent protein; UPR: unfolded protein response.
Assuntos
Autofagia , Schizosaccharomyces , Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3' UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.
Assuntos
Neurônios Receptores Olfatórios , Animais , Camundongos , Axônios/metabolismo , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese/genética , Bulbo Olfatório , Neurônios Receptores Olfatórios/metabolismo , Terminações Pré-Sinápticas/metabolismoRESUMO
Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.