title: Isolation of Mitochondrial Ribosomes.
abstract: Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.
authors.affiliation: The ARC Centre of Excellence for Translational Photosynthesis, The Australian National University, 134 Linnaeus Way, Acton, Canberra, ACT, 2601, Australia. adam.carroll@anu.edu.au.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Carroll
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D003624:Darkness; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000302:isolation & purification*; D059828:Plant Cells / Q000737:chemistry / Q000378:metabolism; D011205:Povidone / Q000737:chemistry; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D003470:Culture Media; D024101:Mitochondrial Proteins; D012269:Ribosomal Proteins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D000645:Ammonium Sulfate
keywords: Mitochondria; Polysomes; Ribosomes; Subcellular fractionation; Translation; Ultracentrifugation