title: Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.
abstract: Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.
authors.affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.
authors.forename: Domenico
authors.initials: D
authors.lastname: Spadafora
authors.affiliation: Department of Physiology and Cell Biology, 307 University Blvd, University of South Alabama, Mobile, Alabama, 36688, United States of America.
authors.forename: Natalia
authors.initials: N
authors.lastname: Kozhukhar
authors.affiliation: Department of Physiology and Cell Biology, 307 University Blvd, University of South Alabama, Mobile, Alabama, 36688, United States of America.|Center for Lung Biology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.
authors.forename: Mikhail F
authors.initials: MF
authors.lastname: Alexeyev
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D056915:DNA Copy Number Variations; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins
chemical_list: D004272:DNA, Mitochondrial; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D029867:Xenopus Proteins; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human; C000618818:Lig3 protein, mouse