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Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

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.

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Effects of DNA ligase activity and of the different pathways for mitochondrial import of the LigA on mtDNA copy number.A, transduction with WT, but not catalytically inactive mLig3 restores mtDNA copy number in clones dependent on LigA for mtDNA replication. B, Intracellular levels of the LigA transcript do not directly correlate with mtDNA copy number. C, LigA targeting to mitochondria through the canonical presequence-dependent pathway restores mtDNA copy number in cells dependent on the non-canonical pathway for LigA import. FRT/FLPo recombination-mediated removal of the MTS attached to LigA leads to the reduction in mtDNA copy number. D, Effects of LigA mitochondrial import through different pathways on mtDNA copy number. mtDNA copy number in original 4B6 cells; in clone #2, which lacks Lig3 and supports mtDNA replication by LigA import through the non-canonical pathway; in clone #2, which was complemented with another copy of the LigA targeted to mitochondria through the canonical pathway; and in complemented LigA clone after recombination-mediated removal of the MTS, which ablates LigA uptake through the canonical pathway.
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pone.0152705.g003: Effects of DNA ligase activity and of the different pathways for mitochondrial import of the LigA on mtDNA copy number.A, transduction with WT, but not catalytically inactive mLig3 restores mtDNA copy number in clones dependent on LigA for mtDNA replication. B, Intracellular levels of the LigA transcript do not directly correlate with mtDNA copy number. C, LigA targeting to mitochondria through the canonical presequence-dependent pathway restores mtDNA copy number in cells dependent on the non-canonical pathway for LigA import. FRT/FLPo recombination-mediated removal of the MTS attached to LigA leads to the reduction in mtDNA copy number. D, Effects of LigA mitochondrial import through different pathways on mtDNA copy number. mtDNA copy number in original 4B6 cells; in clone #2, which lacks Lig3 and supports mtDNA replication by LigA import through the non-canonical pathway; in clone #2, which was complemented with another copy of the LigA targeted to mitochondria through the canonical pathway; and in complemented LigA clone after recombination-mediated removal of the MTS, which ablates LigA uptake through the canonical pathway.

Mentions: To test whether reduced mtDNA copy number in cells with substitution of the LigA for Lig3 depends on the activity of DNA ligase, we used retroviral transduction to deliver either wild type (WT) or catalytically inactive K510V mutant of the mouse Lig3 (S1 Fig) into WT 4B6 cells and into clones with substitution of the LigA for the Lig3. Transduction with WT mLig3 complemented clones with reduced mtDNA copy number, but had little effect on mtDNA content in clone #1, which has high mtDNA content. In contrast, transduction with catalytically inactive mLig3 had little effect on mtDNA content (Fig 3A). Therefore, reduced mitochondrial content of DNA ligase may be directly responsible for reduced mtDNA copy number.


Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

Effects of DNA ligase activity and of the different pathways for mitochondrial import of the LigA on mtDNA copy number.A, transduction with WT, but not catalytically inactive mLig3 restores mtDNA copy number in clones dependent on LigA for mtDNA replication. B, Intracellular levels of the LigA transcript do not directly correlate with mtDNA copy number. C, LigA targeting to mitochondria through the canonical presequence-dependent pathway restores mtDNA copy number in cells dependent on the non-canonical pathway for LigA import. FRT/FLPo recombination-mediated removal of the MTS attached to LigA leads to the reduction in mtDNA copy number. D, Effects of LigA mitochondrial import through different pathways on mtDNA copy number. mtDNA copy number in original 4B6 cells; in clone #2, which lacks Lig3 and supports mtDNA replication by LigA import through the non-canonical pathway; in clone #2, which was complemented with another copy of the LigA targeted to mitochondria through the canonical pathway; and in complemented LigA clone after recombination-mediated removal of the MTS, which ablates LigA uptake through the canonical pathway.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816344&req=5

pone.0152705.g003: Effects of DNA ligase activity and of the different pathways for mitochondrial import of the LigA on mtDNA copy number.A, transduction with WT, but not catalytically inactive mLig3 restores mtDNA copy number in clones dependent on LigA for mtDNA replication. B, Intracellular levels of the LigA transcript do not directly correlate with mtDNA copy number. C, LigA targeting to mitochondria through the canonical presequence-dependent pathway restores mtDNA copy number in cells dependent on the non-canonical pathway for LigA import. FRT/FLPo recombination-mediated removal of the MTS attached to LigA leads to the reduction in mtDNA copy number. D, Effects of LigA mitochondrial import through different pathways on mtDNA copy number. mtDNA copy number in original 4B6 cells; in clone #2, which lacks Lig3 and supports mtDNA replication by LigA import through the non-canonical pathway; in clone #2, which was complemented with another copy of the LigA targeted to mitochondria through the canonical pathway; and in complemented LigA clone after recombination-mediated removal of the MTS, which ablates LigA uptake through the canonical pathway.
Mentions: To test whether reduced mtDNA copy number in cells with substitution of the LigA for Lig3 depends on the activity of DNA ligase, we used retroviral transduction to deliver either wild type (WT) or catalytically inactive K510V mutant of the mouse Lig3 (S1 Fig) into WT 4B6 cells and into clones with substitution of the LigA for the Lig3. Transduction with WT mLig3 complemented clones with reduced mtDNA copy number, but had little effect on mtDNA content in clone #1, which has high mtDNA content. In contrast, transduction with catalytically inactive mLig3 had little effect on mtDNA content (Fig 3A). Therefore, reduced mitochondrial content of DNA ligase may be directly responsible for reduced mtDNA copy number.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

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.

Show MeSH
Related in: MedlinePlus