Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification. 2018

Pin-Chao Liao, and Istvan R Boldogh, and Stephanie E Siegmund, and Zachary Freyberg, and Liza A Pon
Department of Pathology and Cell Biology, Columbia University, New York, NY, United States of America.

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods.

UI MeSH Term Description Entries
D008280 Magnetics The study of MAGNETIC PHENOMENA. Magnetic
D008928 Mitochondria Semiautonomous, self-reproducing organelles that occur in the cytoplasm of all cells of most, but not all, eukaryotes. Each mitochondrion is surrounded by a double limiting membrane. The inner membrane is highly invaginated, and its projections are called cristae. Mitochondria are the sites of the reactions of oxidative phosphorylation, which result in the formation of ATP. They contain distinctive RIBOSOMES, transfer RNAs (RNA, TRANSFER); AMINO ACYL T RNA SYNTHETASES; and elongation and termination factors. Mitochondria depend upon genes within the nucleus of the cells in which they reside for many essential messenger RNAs (RNA, MESSENGER). Mitochondria are believed to have arisen from aerobic bacteria that established a symbiotic relationship with primitive protoeukaryotes. (King & Stansfield, A Dictionary of Genetics, 4th ed) Mitochondrial Contraction,Mitochondrion,Contraction, Mitochondrial,Contractions, Mitochondrial,Mitochondrial Contractions
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D003576 Electron Transport Complex IV A multisubunit enzyme complex containing CYTOCHROME A GROUP; CYTOCHROME A3; two copper atoms; and 13 different protein subunits. It is the terminal oxidase complex of the RESPIRATORY CHAIN and collects electrons that are transferred from the reduced CYTOCHROME C GROUP and donates them to molecular OXYGEN, which is then reduced to water. The redox reaction is simultaneously coupled to the transport of PROTONS across the inner mitochondrial membrane. Cytochrome Oxidase,Cytochrome aa3,Cytochrome-c Oxidase,Cytochrome Oxidase Subunit III,Cytochrome a,a3,Cytochrome c Oxidase Subunit VIa,Cytochrome-c Oxidase (Complex IV),Cytochrome-c Oxidase Subunit III,Cytochrome-c Oxidase Subunit IV,Ferrocytochrome c Oxygen Oxidoreductase,Heme aa3 Cytochrome Oxidase,Pre-CTOX p25,Signal Peptide p25-Subunit IV Cytochrome Oxidase,Subunit III, Cytochrome Oxidase,p25 Presequence Peptide-Cytochrome Oxidase,Cytochrome c Oxidase,Cytochrome c Oxidase Subunit III,Cytochrome c Oxidase Subunit IV,Oxidase, Cytochrome,Oxidase, Cytochrome-c,Signal Peptide p25 Subunit IV Cytochrome Oxidase,p25 Presequence Peptide Cytochrome Oxidase
D006639 Histidine An essential amino acid that is required for the production of HISTAMINE. Histidine, L-isomer,L-Histidine,Histidine, L isomer,L-isomer Histidine
D000090622 Mitochondrial Precursor Protein Import Complex Proteins Multiprotein complexes that mediate import and sorting of mitochondrial preproteins into one of the four mitochondrial subcompartments: the outer membrane, the intermembrane space, the inner membrane, and the matrix. Mitochondrial protein import machinery includes translocase of the outer membrane (TOM) complex, translocases of the inner membrane (TIM) complex, beta‐barrel pathway complex, the mitochondrial intermembrane space import and assembly (MIA) pathway complex, carrier proteins of the inner membrane and the TIM23 complex and sorting and assembly machinery (SAM) complex. MIA Complex,MIM Complex,Mitochondrial Import Complex,Mitochondrial Intermembrane Space Import and Assembly Complex,Mitochondrial Precursor Protein Import Machinery Proteins,Mitochondrial Preprotein Translocases,Mitochondrial Protein Import Machinery Proteins,SAM Complex,Sorting and Assembly Machinery Complex,TIM Complex,TIM23 Complex,TIMM Complex,TOM Channel,TOM Complex,TOM Translocase,TOMM Complex,TOMM Machinery,Translocase Outer Mitochondrial Membrane,Translocase of the Inner Membrane Complex,Translocase of the Inner Mitochondrial Membrane Complex,Translocase of the Outer Membrane Complex,Translocases of the Inner Membrane 23 Complex,Translocases of the Inner Membrane Complex,Import Complex, Mitochondrial,Preprotein Translocases, Mitochondrial,Translocase, TOM,Translocases, Mitochondrial Preprotein
D012441 Saccharomyces cerevisiae A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement. Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D020285 Cryoelectron Microscopy Electron microscopy involving rapid freezing of the samples. The imaging of frozen-hydrated molecules and organelles permits the best possible resolution closest to the living state, free of chemical fixatives or stains. Electron Cryomicroscopy,Cryo-electron Microscopy,Cryo electron Microscopy,Cryo-electron Microscopies,Cryoelectron Microscopies,Cryomicroscopies, Electron,Cryomicroscopy, Electron,Electron Cryomicroscopies,Microscopies, Cryo-electron,Microscopies, Cryoelectron,Microscopy, Cryo-electron,Microscopy, Cryoelectron
D024101 Mitochondrial Proteins Proteins encoded by the mitochondrial genome or proteins encoded by the nuclear genome that are imported to and resident in the MITOCHONDRIA. Proteins, Mitochondrial,Mitochondrial Protein,Protein, Mitochondrial

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