Abstract
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.
Highlights
Isolated yeast mitochondria are used to study fundamental processes including mitochondrial respiration, metabolic activity, protein import and membrane fusion as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs and other organelles
We find that proteinase K treatment decreased Tom70 levels but not cytochrome b2 (Cyb2) levels in crude and magnetic bead-purified mitochondria (Fig 5A and 5B)
In mitochondria treated with valinomycin, which abolishes mitochondrial Δψ, only full-length Su9-DHFR is associated with the organelle (Fig 5C, Lane 4 and 7). These findings indicate that mitochondria prepared using either method have mitochondrial protein import activity: they bind to Su9-DHFR and exhibit Δψ-dependent translocation of the protein across mitochondrial membranes and processing of Su9-DHFR to its mature form [1, 27, 28]
Summary
Isolated yeast mitochondria are used to study fundamental processes including mitochondrial respiration, metabolic activity, protein import and membrane fusion as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs and other organelles. Isolated mitochondria are utilized to investigate the assembly and interactions of mitochondrial protein complexes [1,2,3,4,5,6,7,8]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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