Spadafora D et al. (2016), Presequence-Independent Mitochondrial Import of...

XB-ART-52849

PLoS One 2016 Mar 31;113:e0152705. doi: 10.1371/journal.pone.0152705.

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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 .

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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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Species referenced: Xenopus
Genes referenced: lig3 myc

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	Fig 1. The effect of LigA on mtDNA copy number in 4B6 cells.A, 4B6 cells were transduced with a retrovirus encoding LigA without MTS, and mtDNA copy number was determined in four resulting subclones. B, Subclone #11 was transduced with a retrovirus encoding Cre recombinase, and genomic DNA from ten clones was PCR-analyzed for the presence of unexcised Lig3 allele (Lig3), excised Lig3 allele (ΔLig3), ρ0 phenotype (mtDNA and nDNA primers), and for the presence of LigA (LigA). Primer sets and primer sequences are listed in the S1 Table.
	Fig 2. Stability of mtDNA inheritance.Clone #3 was re-cloned in either selective medium devoid of uridine and pyruvate (-UP) or in non-selective medium containing uridine and pyruvate (+UP) after 3 weeks of growth in non-selective medium. Subclones grown in either selective (A) or non-selective (B) medium were tested for the presence of mtDNA by PCR. C. mtDNA copy number was determined in 5 clones grown in either selective (-UP) or non-selective (+UP) conditions, and means were compared (D). ns, not significant, two-tailed Student’s t-test assuming unequal variance. E. mtDNA copy number was determined in six selected clones after 20-day propagation in either selective (-UP) or non-selective (+UP) media.
	Fig 3. 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.
	Fig 4. Respiration and growth rates.4B6 cells and cloned with LigA-supported mtDNA replication #1,#2, and #4 were transduced with retroviruses encoding either WT or catalytically inactive K510V mutant mLig3. A-C, OCR was determined in the parental (A) and transduced (B and C) cells. D, doubling time of the resulting clones. ***, P<0.001, two-way ANOVA with Dunnett’s post-hoc test.
	Fig 5. ChVlig can support mtDNA replication at reduced copy number.A, 4B6 cells were sequentially transduced with retroviruses encoding ChVLigAnd Cre recombinase, and Lig3 excision was tested in 13 resulting clones. B, resulting clones have variable mtDNA copy number as compared to an arbitrarily chosen clone#1. C, Clones #5, #6, and #13 maintained >90% reduced mtDNA copy number upon 3-week propagation in the selective medium. D, mtDNA copy number in the 4B6 cells transduced with ChVlig, two clones (#3 and #14), which were derived from the original ChVlig-transduced clone by Lig3 excision, and in the clone#14 after transduction with either WT or catalytically inactive mLig3.
	Fig 6. Presequence-independent mitochondrial import of LigA-myc is inefficient.A. Genotyping cell lines transduced with either MTS-LigA-myc or with LigA-myc; B. mtDNA content in the parental 4B6 cells, and in cells in which mtDNA replication is supported by either MTS-LigA-myc (+MTS) or LigA-myc (No MTS); C. Whole cell and mitochondrial fractions from cells that express either MTS-LigA-myc or LigA-myc were subjected to western blotting with myc-tag antibodies, or with antibodies against reference proteins TOM70 (resides in the mitochondrial outer membrane) or MnSOD (resides in the mitochondrial matrix).
	Fig 7. A method for establishing mouse cell lines with reduced mtDNA copy number.A and B, initial screening for mtDNA copy number in 3T3#53 clones expressing LigA, in which Lig3 exon 1 or exon 8 was targeted with CRISPR-Cas9. C and D, The relationship between Lig3 inactivation and mtDNA copy number in clones with targeted exon 1 and exon 8.

References [+] :

Acín-Pérez, Respiratory complex III is required to maintain complex I in mammalian mitochondria. 2004, Pubmed