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Mitochondrial proteome analysis during MceC expression time-course

ABSTRACT: Mitochondria were isolated from tetracycline-inducible control (EV) HEK293 cells or HEK293 cells expressing the C. burnetii effector protein MceC for 1, 4 and 8 hours.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Kidney Cell

DISEASE(S): Q Fever

SUBMITTER: Nichollas Scott

LAB HEAD: Hayley J Newton

PROVIDER: PXD019337 | Pride | 2020-11-12

REPOSITORIES: Pride

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			Action	DRS
	200320_Laura_LF_496_1.raw	Raw
	200320_Laura_LF_496_10.raw	Raw
	200320_Laura_LF_496_11.raw	Raw
	200320_Laura_LF_496_12.raw	Raw
	200320_Laura_LF_496_13.raw	Raw

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Quantitative mitochondrial proteome analysis during MceC expression time-course

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Project description:Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it remains highly controversial if POLRMT also serves as the primase for initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion and TFAM is thus protected from degradation of the AAA+ Lon protease in absence of POLRMT. Lastly, mitochondrial transcription elongation factor (TEFM) can compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role for this factor in transcription. In conclusion, we present here the first in vivo evidence that POLRMT has a key regulatory role in replication of mammalian mtDNA and is part of a mechanism that provides a switch between RNA primer formation for mtDNA replication and mtDNA expression. Isolated heart mitochondria from three control mice (L/L) and three Polrmt knockout mice (L/L, cre), aged 3-4 weeks, were sequenced and analyzed for differential expression.

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