RESUMEN
Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery, the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation.
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Autofagia , Células Eucariotas/citología , Redes y Vías Metabólicas , Animales , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Células Eucariotas/clasificación , Células Eucariotas/patología , Humanos , Mitocondrias/patología , FosforilaciónRESUMEN
Protein scaffolds coordinate the assembly of many multicomponent signaling complexes. Bodemann et al. (2011) now show that the exocyst, a protein complex involved in tethering transport vesicles to the plasma membrane, provides an assembly and activation platform for components of the autophagy machinery via a process requiring the GTPase RalB.
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Despite major advances in our understanding of players and mechanisms involved in peroxisome biogenesis and peroxisome degradation, very few studies have focused on unraveling the multi-layered connections between, and the coordination of, these two opposing processes that regulate peroxisome homeostasis. The intersection between these processes also provides exciting avenues for future research. This review highlights the links between peroxisome biogenesis and degradation, incorporating an integrative approach that is critical not only for a mechanistic understanding, but also for manipulating the balance between these processes in relevant disease models.
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Redes y Vías Metabólicas , Peroxisomas , Homeostasis , Peroxisomas/metabolismoRESUMEN
SUMMARY: perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. AVAILABILITY AND IMPLEMENTATION: The software is coded in Python. Compiled executables and source code are available at https://github.com/AitchisonLab/perox-per-cell. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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We have developed a general progressive procedure, Active Interaction Mapping, to guide assembly of the hierarchy of functions encoding any biological system. Using this process, we assemble an ontology of functions comprising autophagy, a central recycling process implicated in numerous diseases. A first-generation model, built from existing gene networks in Saccharomyces, captures most known autophagy components in broad relation to vesicle transport, cell cycle, and stress response. Systematic analysis identifies synthetic-lethal interactions as most informative for further experiments; consequently, we saturate the model with 156,364 such measurements across autophagy-activating conditions. These targeted interactions provide more information about autophagy than all previous datasets, producing a second-generation ontology of 220 functions. Approximately half are previously unknown; we confirm roles for Gyp1 at the phagophore-assembly site, Atg24 in cargo engulfment, Atg26 in cytoplasm-to-vacuole targeting, and Ssd1, Did4, and others in selective and non-selective autophagy. The procedure and autophagy hierarchy are at http://atgo.ucsd.edu/.
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Autofagia/genética , Redes Reguladoras de Genes , Genómica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Biología de Sistemas/métodos , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Bases de Datos Genéticas , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Humanos , Modelos Genéticos , Pichia/genética , Pichia/metabolismo , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Integración de SistemasRESUMEN
BACKGROUND: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution. RESULTS: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role. CONCLUSION: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.
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Anopheles/genética , Expresión Génica , Genoma , Resistencia a los Insecticidas/genética , Mosquitos Vectores/genética , Animales , Femenino , Perfilación de la Expresión Génica , Malaria/transmisión , MasculinoRESUMEN
Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.
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Retículo Endoplásmico/genética , Proteínas de la Membrana/biosíntesis , Mitocondrias/genética , Peroxisomas/genética , Retículo Endoplásmico/metabolismo , Células Eucariotas/metabolismo , Homeostasis/genética , Humanos , Proteínas de la Membrana/genética , Redes y Vías Metabólicas/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Oxidación-Reducción , Peroxisomas/metabolismoRESUMEN
The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.
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Proteínas Fúngicas/metabolismo , Malato Deshidrogenasa/metabolismo , Ácido Oléico/metabolismo , Peroxisomas/metabolismo , Saccharomycetales/enzimología , Carbono/farmacología , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , NAD/metabolismo , Transporte de Proteínas/efectos de los fármacos , Reproducibilidad de los ResultadosRESUMEN
The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division, and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted the direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-containing PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10 while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reversed the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3- and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.
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Proteínas Fúngicas , Membranas Intracelulares , Proteínas de la Membrana , Peroxisomas , Pichia , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Peroxisomas/química , Peroxisomas/genética , Peroxisomas/metabolismo , Pichia/química , Pichia/genética , Pichia/metabolismo , Eliminación de SecuenciaRESUMEN
Peroxisomes proliferate by growth and division of pre-existing peroxisomes or could arise de novo. Though the de novo pathway of peroxisome biogenesis is a more recent discovery, several studies have highlighted key mechanistic details of the pathway. The endoplasmic reticulum (ER) is the primary source of lipids and proteins for the newly-formed peroxisomes. More recently, an intricate sorting process functioning at the ER has been proposed, that segregates specific PMPs first to peroxisome-specific ER domains (pER) and then assembles PMPs selectively into distinct pre-peroxisomal vesicles (ppVs) that later fuse to form import-competent peroxisomes. In addition, plausible roles of the three key peroxins Pex3, Pex16 and Pex19, which are also central to the growth and division pathway, have been suggested in the de novo process. In this review, we discuss key developments and highlight the unexplored avenues in de novo peroxisome biogenesis.
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Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Biogénesis de Organelos , Peroxisomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Retículo Endoplásmico/química , Células Eucariotas/química , Células Eucariotas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Peroxinas , Peroxisomas/química , Plantas/química , Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Levaduras/química , Levaduras/metabolismoRESUMEN
Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.
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Autofagia , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Pichia/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de ProteínasRESUMEN
Peroxisomes are essential organelles required for proper cell function in all eukaryotic organisms. They participate in a wide range of cellular processes including the metabolism of lipids and generation, as well as detoxification, of hydrogen peroxide (H2O2). Therefore, peroxisome homoeostasis, manifested by the precise and efficient control of peroxisome number and functionality, must be tightly regulated in response to environmental changes. Due to the existence of many physiological disorders and diseases associated with peroxisome homoeostasis imbalance, the dynamics of peroxisomes have been widely examined. The increasing volume of reports demonstrating significant involvement of the autophagy machinery in peroxisome removal leads us to summarize current knowledge of peroxisome degradation in mammalian cells. In this review we present current models of peroxisome degradation. We particularly focus on pexophagy-the selective clearance of peroxisomes through autophagy. We also critically discuss concepts of peroxisome recognition for pexophagy, including signalling and selectivity factors. Finally, we present examples of the pathological effects of pexophagy dysfunction and suggest promising future directions.
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Autofagia , Peroxisomas/metabolismo , Animales , Humanos , Mamíferos , Modelos BiológicosRESUMEN
Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming ß-hemolysin/cytolysin (ß-h/c) trigger autophagic activation. Cell-free bacterial extracts containing ß-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a ß-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.
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Autofagia , Barrera Hematoencefálica/microbiología , Células Endoteliales/fisiología , Meningitis Neumocócica/patología , Streptococcus agalactiae/fisiología , Animales , Toxinas Bacterianas/biosíntesis , Barrera Hematoencefálica/patología , Células Cultivadas , Células Endoteliales/microbiología , Endotelio Vascular/microbiología , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Transporte de ProteínasRESUMEN
Human hepatitis B virus (HBV) causes chronic hepatitis and is associated with the development of hepatocellular carcinoma. HBV infection alters mitochondrial metabolism. The selective removal of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. Here, we report that HBV shifts the balance of mitochondrial dynamics toward fission and mitophagy to attenuate the virus-induced apoptosis. HBV induced perinuclear clustering of mitochondria and triggered mitochondrial translocation of the dynamin-related protein (Drp1) by stimulating its phosphorylation at Ser616, leading to mitochondrial fission. HBV also stimulated the gene expression of Parkin, PINK1, and LC3B and induced Parkin recruitment to the mitochondria. Upon translocation to mitochondria, Parkin, an E3 ubiquitin ligase, underwent self-ubiquitination and facilitated the ubiquitination and degradation of its substrate Mitofusin 2 (Mfn2), a mediator of mitochondrial fusion. In addition to conventional immunofluorescence, a sensitive dual fluorescence reporter expressing mito-mRFP-EGFP fused in-frame to a mitochondrial targeting sequence was employed to observe the completion of the mitophagic process by delivery of the engulfed mitochondria to lysosomes for degradation. Furthermore, we demonstrate that viral HBx protein plays a central role in promoting aberrant mitochondrial dynamics either when expressed alone or in the context of viral genome. Perturbing mitophagy by silencing Parkin led to enhanced apoptotic signaling, suggesting that HBV-induced mitochondrial fission and mitophagy promote cell survival and possibly viral persistence. Altered mitochondrial dynamics associated with HBV infection may contribute to mitochondrial injury and liver disease pathogenesis.
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Apoptosis , Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Dinámicas Mitocondriales , Mitofagia , Apoptosis/genética , Células Cultivadas , Dinaminas , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Células Hep G2 , Hepatitis B/genética , Hepatitis B/patología , Virus de la Hepatitis B/patogenicidad , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia/genética , Fagosomas/virología , Transporte de Proteínas , Transactivadores/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Autophagy and autophagy-related processes are fundamentally important in human health and disease. These processes are viewed primarily as cellular degradative pathways that recycle macromolecules and dysfunctional or redundant organelles into amino acids, sugars and lipids, especially during starvation. However, the ubiquitin-like autophagy proteins and other components of the autophagic machinery additionally participate in cellular reprogramming. We highlight these non-autophagic roles of autophagy proteins with the aim of drawing attention to this growing, but unexplored, research topic. We focus on the non-autophagic functions of autophagy proteins in cell survival and apoptosis, modulation of cellular traffic, protein secretion, cell signalling, transcription, translation and membrane reorganization.
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Autofagia , Péptidos y Proteínas de Señalización Intracelular/fisiología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/fisiología , Adipogénesis , Animales , Proteína 12 Relacionada con la Autofagia , Proteína 5 Relacionada con la Autofagia , Proteína 7 Relacionada con la Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Exocitosis , Interacciones Huésped-Patógeno , Humanos , Proteínas de Microfilamentos/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Transporte de Proteínas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Enzimas Activadoras de Ubiquitina/fisiologíaRESUMEN
The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin-like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8-interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.
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Proteínas Asociadas a Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Sitios de Unión , Secuencia de Consenso , Técnicas de Inactivación de Genes , Mitofagia , Datos de Secuencia Molecular , Fosforilación , Pichia/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The knowledge on protein-protein interactions (PPI) and their related pathways are equally important to understand the biological functions of the living cell. Such information on human proteins is highly desirable to understand the mechanism of several diseases such as cancer, diabetes, and Alzheimer's disease. Because much of that information is buried in biomedical literature, an automated text mining system for visualizing human PPI and pathways is highly desirable. In this paper, we present HPIminer, a text mining system for visualizing human protein interactions and pathways from biomedical literature. HPIminer extracts human PPI information and PPI pairs from biomedical literature, and visualize their associated interactions, networks and pathways using two curated databases HPRD and KEGG. To our knowledge, HPIminer is the first system to build interaction networks from literature as well as curated databases. Further, the new interactions mined only from literature and not reported earlier in databases are highlighted as new. A comparative study with other similar tools shows that the resultant network is more informative and provides additional information on interacting proteins and their associated networks.
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Biología Computacional/métodos , Minería de Datos/métodos , Mapeo de Interacción de Proteínas/métodos , Enfermedad de Alzheimer/metabolismo , Gráficos por Computador , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos Teóricos , Interfaz Usuario-ComputadorRESUMEN
In Pichia pastoris, the peroxisomal targeting signal 2 (PTS2)-dependent peroxisomal matrix protein import pathway requires the receptor, Pex7, and its co-receptor Pex20. A conserved lysine (Lys(19)) near the N terminus of Pex20 is required for its polyubiquitination and proteasomal degradation, whereas a conserved cysteine (Cys(8)) is essential for its recycling. In this study, we found that Cys(8) is required for the DTT-sensitive mono- and diubiquitination of Pex20. We also show that the PTS2 cargo receptor, Pex7, is required for Pex20 polyubiquitination. Pex4, the E2 ubiquitin-conjugation enzyme, is required for monoubiquitination of Pex20. However, it is also necessary for polyubiquitination of Pex20, making its behavior distinct from the ubiquitination described for other PTS receptors. Unlike the roles of specific RING peroxins in Pex5 ubiquitination, we found that all the RING peroxins (Pex2, Pex10, and Pex12) are required as E3 ubiquitin ligases for Pex20 mono- and polyubiquitination. A model for Pex20 ubiquitination is proposed based on these observations. This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this PTS2 cargo co-receptor.
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Proteínas Fúngicas/metabolismo , Pichia/metabolismo , Poliubiquitina/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Sitios de Unión/genética , Cisteína/genética , Cisteína/metabolismo , Ditiotreitol/farmacología , Proteínas Fúngicas/genética , Immunoblotting , Lisina/genética , Lisina/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Mutación , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Peroxisomas/metabolismo , Pichia/genética , Regiones Promotoras Genéticas/genética , Dominios RING Finger/genética , Receptores Citoplasmáticos y Nucleares/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.