Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Abstract

Lon is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. Although a role for Lon in mitochondrial biogenesis has been proposed, the mechanistic basis is unclear. Here, we demonstrate a role for Lon in mtDNA metabolism. An RNA interference (RNAi) construct was designed that reduces Lon to less than 10% of its normal level in Drosophila Schneider cells. RNAi knockdown of Lon results in increased abundance of mitochondrial transcription factor A (TFAM) and mtDNA copy number. In a corollary manner, overexpression of Lon reduces TFAM levels and mtDNA copy number. Notably, induction of mtDNA depletion in Lon knockdown cells does not result in degradation of TFAM, thereby causing a dramatic increase in the TFAMmtDNA ratio. The increased TFAMmtDNA ratio in turn causes inhibition of mitochondrial transcription. We conclude that Lon regulates mitochondrial transcription by stabilizing the mitochondrial TFAMmtDNA ratio via selective degradation of TFAM.

Fig. 1.

Expression of Drosophila Lon protease in Schneider cells. Schneider cells with no plasmid (control) or carrying pMt/Hy (vector) or pMt/Lon/Hy (Lon WT) were cultured for 10 d in the presence or absence of 0.2 mM CuSO4. (A) Protein extracts (20 μg) were fractionated by 7.5%, 10.5%, or 13.5% SDS-PAGE, transferred to nitrocellulose filters and probed with antibodies against Lon protease, TFAM, mtTFB2, mtDNA helicase, pol γ-α, mtSSB, ATPase β, or β tubulin as indicated. (B) The TFAM/ β tubulin ratio was quantitated by normalizing TFAM protein levels to β tubulin protein levels as described under Materials and Methods. Error bars indicate means ± standard error of three independent experiments. The asterisk indicates P < 0.05 in comparison to control. (C) Total DNA (10 μg) was extracted from Schneider cells or Schneider cells carrying pMt/Hy or pMt/Lon/Hy that were cultured for 10 d in the presence of 0.2 mM CuSO4. DNA was digested with XhoI, fractionated in a 0.7% agarose/ TBE gel, and then blotted to a nylon membrane. The membrane was hybridized with a radiolabeled probe for CytB, and then stripped and rehybridized with radiolabeled probe for the histone gene cluster as a control. The relative mtDNA copy number was quantitated as described under Materials and Methods. Error bars indicate means ± standard error of three independent experiments. The asterisk indicates P < 0.05 in comparison to control. (D) Total RNA (10 μg) was extracted from Schneider cells or Schneider cells carrying pMt/Hy or pMt/Lon/Hy after 10 d of culture in the presence or absence of 0.2 mM CuSO4. RNA was fractionated in a 1.2% agarose/formaldehyde gel, blotted to nylon membrane, and hybridized with radiolabeled probes for the mitochondrial transcripts 12S rRNA, ND4, and Cytb, the nuclear transcript RP49, and TFAM.

Fig. 2.

Expression of Drosophila Lon-targeted RNAi in Schneider cells. Schneider cells with no plasmid (control) or carrying pMt/Hy (vector) or pMt/invLon/Hy (Lon RNAi) were cultured for 10 d in the presence or absence of 0.2 mM CuSO4. (A) Immunoblot analysis was carried out as described in the legend to Fig. 1A. (B) The TFAM/β tubulin ratio was quantitated by normalizing TFAM protein levels to β tubulin protein levels as described under Materials and Methods. Error bars indicate means ± standard error of three independent experiments. The asterisk indicates P < 0.05 in comparison to control. (C) Relative mtDNA copy number was determined as described in the figure legend to Fig. 1C. Error bars indicate means ± standard error of three independent experiments. The asterisk indicates P < 0.05 in comparison to control. (D) Northern blot analysis using 12S rRNA, ND4, Cytb, RP49, and TFAM was carried out as described in the legend to Fig. 1D.

Fig. 3.

Dynamics of mitochondrial nucleoid proteins during mtDNA depletion in Lon overexpressing or knockdown Schneider cells. Schneider cells with no plasmid (control) or carrying pMt/Lon/Hy (Lon WT) or pMt/invLon/Hy (Lon RNAi) were cultured for 6 d in the presence of 200 ng/mL EtBr. The cells were harvested prior to EtBr treatment (0 d) and after 2, 4, and 6 days of EtBr treatment. (A) Immunoblot analysis was carried out as described in the legend to Fig. 1A. (B) Relative mtDNA copy number was determined as described in the legend to Fig. 1C. Error bars indicate means ± standard error of two independent experiments. (C) The TFAM/ mtDNA ratio was quantitated by normalizing TFAM levels to relative mtDNA copy number. Error bars indicate means ± standard error of two independent experiments.

Fig. 4.

Expression of TFAM in Schneider cells. Relative ratio of mtDNA copy number (open circles, solid line), mt mRNAs/nRNA (crosses, dotted line), and TFAM/mtDNA (filled triangles, dashed line) were measured at different overexpression levels of TFAM in Schneider cells as indicated. Relative mtDNA copy number was determined as described in the legend to Fig. 1C. The mt mRNAs/nRNA ratio was quantitated by normalizing mitochondrial transcript abundance (ND4 and Cyt b) to that of nuclear Rp49. TFAM/mtDNA ratio was quantitated by normalizing TFAM protein levels to relative mtDNA copy number.

Fig. 5.

Effects of Lon knockdown on mitochondrial transcript levels after mtDNA depletion in Schneider cells. Schneider cells carrying no plasmid (control) and or pMt/invLon/Hy (Lon RNAi) were cultured for 10 d in the presence or absence of dsRNA of mtDNA helicase, pol γ-α, or GFP as control. (A) Immunoblot analysis was carried out as described in the legend to Fig. 1A. (B) Relative mtDNA copy number was determined as described in the legend to Fig. 1C. Error bars indicate means ± standard error of two independent experiments. The asterisk indicates P < 0.05 in comparison to the control. (C) The ratio of TFAM/mtDNA was determined as described in the legend to Fig. 3C. Error bars indicate means ± standard error of two independent experiments. The asterisk indicates P < 0.05 in comparison to each cell that was cultured in the absence of dsRNA. (D) Northern blot analysis using ND4, Cytb, and RP49 was carried out as described in the legend to Fig. 1D. Relative mitochondrial mRNA levels were quantitated by normalizing ND4 and Cytb abundance to that of RP49. Error bars indicate means ± SE of two independent experiments. The asterisk indicates P < 0.05 in comparison to the control.