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1.
Nucleic Acids Res ; 49(D1): D1541-D1547, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33174596

RESUMEN

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.


Asunto(s)
Bases de Datos de Proteínas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Anotación de Secuencia Molecular , Proteoma/metabolismo , Animales , Teorema de Bayes , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Conjuntos de Datos como Asunto , Humanos , Internet , Aprendizaje Automático , Espectrometría de Masas , Ratones , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/genética , Proteoma/clasificación , Proteoma/genética , Programas Informáticos
2.
Nat Methods ; 12(1): 51-4, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25419960

RESUMEN

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.


Asunto(s)
Ascorbato Peroxidasas/biosíntesis , Microscopía Electrónica de Transmisión/métodos , Proteómica/métodos , Animales , Ascorbato Peroxidasas/genética , Células COS , Proteínas de Unión al Calcio/análisis , Proteínas de Transporte de Catión/análisis , Chlorocebus aethiops , Evolución Molecular Dirigida/métodos , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/análisis , Saccharomyces cerevisiae/enzimología
3.
Nat Protoc ; 12(9): 1792-1816, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28796234

RESUMEN

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).


Asunto(s)
Ascorbato Peroxidasas/genética , Técnicas Citológicas/métodos , Técnicas Genéticas , Microscopía Electrónica/métodos , Imagen Molecular/métodos , Animales , Células COS , Células Cultivadas , Estructuras Celulares/ultraestructura , Chlorocebus aethiops , Células HEK293 , Hipocampo/citología , Humanos , Ratas , Proteínas Recombinantes de Fusión/genética
4.
Elife ; 62017 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-28441135

RESUMEN

The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP's ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.


Asunto(s)
Retículo Endoplásmico/química , Membranas Intracelulares/química , Proteínas de la Membrana/análisis , Mitocondrias/química , Proteómica/métodos , Animales , Biotinilación/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Peroxidasa/metabolismo
5.
Nat Protoc ; 11(3): 456-75, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26866790

RESUMEN

This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a 'ratiometric' three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5-7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2-5 d and analysis of the data to obtain the final proteomic list takes 1 week.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteoma/análisis , Proteómica/métodos , Biotina/metabolismo , Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Endonucleasas , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Marcaje Isotópico/métodos , Espectrometría de Masas , Enzimas Multifuncionales , Ingeniería de Proteínas , Proteoma/metabolismo
7.
PLoS One ; 8(2): e52823, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23457442

RESUMEN

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.


Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/análisis , Uniones Intercelulares/metabolismo , Proteínas del Tejido Nervioso/análisis , Neuronas/citología , Mapeo de Interacción de Proteínas/métodos , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/ultraestructura , Animales , Biotina/metabolismo , Biotinilación , Células Cultivadas , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Uniones Intercelulares/ultraestructura , Ligasas/análisis , Ligasas/metabolismo , Microscopía Confocal/métodos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado/métodos
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