title: High throughput gene complementation screening permits identification of a mammalian mitochondrial protein synthesis (ρ(-)) mutant.
abstract: To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14 kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
authors.affiliation: Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Department of Pathology and Laboratory of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Prasanth
authors.initials: P
authors.lastname: Potluri
authors.affiliation: Dépt. de Biochimie et Génétique, Centre Hospitalier Universitaire d'Angers, Angers, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Procaccio
authors.affiliation: Division of Biological Sciences, University of California - San Diego, La Jolla, CA, United States.
authors.forename: Immo E
authors.initials: IE
authors.lastname: Scheffler
authors.affiliation: Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Department of Pathology and Laboratory of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: wallaced1@email.chop.edu.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Wallace
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D005690:Galactose / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D000069396:Mitochondrial Ribosomes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010085:Oxidative Phosphorylation; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D024101:Mitochondrial Proteins; D010455:Peptides; D012335:RNA, Ribosomal; D005690:Galactose
keywords: Complementation cloning; Mitochondrial diseases; Mitochondrial ribosomes; OXPHOS; bS6m