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The functional organization of mitochondrial genomes in human cells.

Iborra FJ, Kimura H, Cook PR - BMC Biol. (2004)

Bottom Line: This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes.Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK. francisco.iborra@imm.ox.ac.uk <francisco.iborra@imm.ox.ac.uk>

ABSTRACT

Background: We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.

Results: Mitochondrial DNA is organized in approximately 475 discrete foci containing 6-10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 x 10(-3) microm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time approximately 43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).

Conclusions: The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

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Localization of the cytoplasmic translation and mitochondrial import machinery relative to newly-made mitochondrial transcripts. Cells were grown in BrU for 30 min, fixed, Br-RNA plus different targets immunolabelled, and confocal images collected. Under the conditions used, little Br-RNA made in the nucleus has time to reach the cytoplasm [39]. (A-C) Three views of one field; newly-made mitochondrial Br-RNA is found near S6, a constituent of the cytoplasmic ribosome (shown as red and green in the merge in (C), respectively). (D) The high Pearson's coefficient (rP) at Δx values close to zero confirms that Br-RNA is often found near S6. (E-L) As (A-D); newly-made mitochondrial Br-RNA is also found near the α subunit of NAC (a cytoplasmic chaperone), and Tom22 (a component of the mitochondrial import machinery). Bar: 1 μm.
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Figure 8: Localization of the cytoplasmic translation and mitochondrial import machinery relative to newly-made mitochondrial transcripts. Cells were grown in BrU for 30 min, fixed, Br-RNA plus different targets immunolabelled, and confocal images collected. Under the conditions used, little Br-RNA made in the nucleus has time to reach the cytoplasm [39]. (A-C) Three views of one field; newly-made mitochondrial Br-RNA is found near S6, a constituent of the cytoplasmic ribosome (shown as red and green in the merge in (C), respectively). (D) The high Pearson's coefficient (rP) at Δx values close to zero confirms that Br-RNA is often found near S6. (E-L) As (A-D); newly-made mitochondrial Br-RNA is also found near the α subunit of NAC (a cytoplasmic chaperone), and Tom22 (a component of the mitochondrial import machinery). Bar: 1 μm.

Mentions: We might expect mitochondrial ribosomes to translate co-transcriptionally like their relatives – the bacterial ribosomes – and because the amino-terminal domain of the mitochondrial RNA (mtRNA) polymerase can interact with the mitochondrial translation machinery [41]. Therefore, we wished to label mitochondrial translation sites. Unfortunately, appropriate antibodies to mitochondrial ribosomes are not available, and the labelled precursors (that is, biotin-lys-tRNA, BODIPY-lys-tRNA) that we use to mark extra-mitochondrial translation sites with high resolution [42] cannot be used here; mitochondria contain high concentrations of biotinylated proteins that provide a high background [43] and BODIPY has a similar emission spectrum to YFP. However, proteins encoded by the mitochondrial genome do complex with others encoded by the nuclear genome to form the mitochondrion, and we reasoned that the different synthetic machineries might lie close together on different sides of the mitochondrial membranes so the proteins they produce could combine together efficiently. Therefore, we localized newly-made mtRNA relative to extra-mitochondrial ribosomes. Cells were grown briefly in BrU, fixed, and the resulting mitochondrial Br-RNA localized relative to S6, an extra-mitochondrial ribosomal protein. Most S6 is found in discrete foci remote from mitochondria; these presumably contain clusters of cytoplasmic ribosomes. However, some foci lie close to mitochondrial foci containing Br-RNA (Figure 8A,8B,8C), and this close association was confirmed by cross-correlation analysis (Figure 8D). Newly-made mtRNA also lies near foci containing nascent peptide associated protein (NAC) (Figure 8E,8F,8G,8H), a complex involved in directing nascent cytoplasmic polypeptides to the appropriate cellular compartment and which is known to associate with mitochondrial membranes [44]. Newly-made mtRNA also colocalizes with Tom 22 (Figure 8I,8J,8K,8L), a component of the machinery that imports nuclear-encoded proteins into mitochondria [11,12]. These results are consistent with an association of the mitochondrial transcription and translation machinery on one side of the mitochondrial membrane with the cytoplasmic translation machinery and import machinery on the other.


The functional organization of mitochondrial genomes in human cells.

Iborra FJ, Kimura H, Cook PR - BMC Biol. (2004)

Localization of the cytoplasmic translation and mitochondrial import machinery relative to newly-made mitochondrial transcripts. Cells were grown in BrU for 30 min, fixed, Br-RNA plus different targets immunolabelled, and confocal images collected. Under the conditions used, little Br-RNA made in the nucleus has time to reach the cytoplasm [39]. (A-C) Three views of one field; newly-made mitochondrial Br-RNA is found near S6, a constituent of the cytoplasmic ribosome (shown as red and green in the merge in (C), respectively). (D) The high Pearson's coefficient (rP) at Δx values close to zero confirms that Br-RNA is often found near S6. (E-L) As (A-D); newly-made mitochondrial Br-RNA is also found near the α subunit of NAC (a cytoplasmic chaperone), and Tom22 (a component of the mitochondrial import machinery). Bar: 1 μm.
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Related In: Results  -  Collection

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Figure 8: Localization of the cytoplasmic translation and mitochondrial import machinery relative to newly-made mitochondrial transcripts. Cells were grown in BrU for 30 min, fixed, Br-RNA plus different targets immunolabelled, and confocal images collected. Under the conditions used, little Br-RNA made in the nucleus has time to reach the cytoplasm [39]. (A-C) Three views of one field; newly-made mitochondrial Br-RNA is found near S6, a constituent of the cytoplasmic ribosome (shown as red and green in the merge in (C), respectively). (D) The high Pearson's coefficient (rP) at Δx values close to zero confirms that Br-RNA is often found near S6. (E-L) As (A-D); newly-made mitochondrial Br-RNA is also found near the α subunit of NAC (a cytoplasmic chaperone), and Tom22 (a component of the mitochondrial import machinery). Bar: 1 μm.
Mentions: We might expect mitochondrial ribosomes to translate co-transcriptionally like their relatives – the bacterial ribosomes – and because the amino-terminal domain of the mitochondrial RNA (mtRNA) polymerase can interact with the mitochondrial translation machinery [41]. Therefore, we wished to label mitochondrial translation sites. Unfortunately, appropriate antibodies to mitochondrial ribosomes are not available, and the labelled precursors (that is, biotin-lys-tRNA, BODIPY-lys-tRNA) that we use to mark extra-mitochondrial translation sites with high resolution [42] cannot be used here; mitochondria contain high concentrations of biotinylated proteins that provide a high background [43] and BODIPY has a similar emission spectrum to YFP. However, proteins encoded by the mitochondrial genome do complex with others encoded by the nuclear genome to form the mitochondrion, and we reasoned that the different synthetic machineries might lie close together on different sides of the mitochondrial membranes so the proteins they produce could combine together efficiently. Therefore, we localized newly-made mtRNA relative to extra-mitochondrial ribosomes. Cells were grown briefly in BrU, fixed, and the resulting mitochondrial Br-RNA localized relative to S6, an extra-mitochondrial ribosomal protein. Most S6 is found in discrete foci remote from mitochondria; these presumably contain clusters of cytoplasmic ribosomes. However, some foci lie close to mitochondrial foci containing Br-RNA (Figure 8A,8B,8C), and this close association was confirmed by cross-correlation analysis (Figure 8D). Newly-made mtRNA also lies near foci containing nascent peptide associated protein (NAC) (Figure 8E,8F,8G,8H), a complex involved in directing nascent cytoplasmic polypeptides to the appropriate cellular compartment and which is known to associate with mitochondrial membranes [44]. Newly-made mtRNA also colocalizes with Tom 22 (Figure 8I,8J,8K,8L), a component of the machinery that imports nuclear-encoded proteins into mitochondria [11,12]. These results are consistent with an association of the mitochondrial transcription and translation machinery on one side of the mitochondrial membrane with the cytoplasmic translation machinery and import machinery on the other.

Bottom Line: This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes.Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK. francisco.iborra@imm.ox.ac.uk <francisco.iborra@imm.ox.ac.uk>

ABSTRACT

Background: We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.

Results: Mitochondrial DNA is organized in approximately 475 discrete foci containing 6-10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 x 10(-3) microm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time approximately 43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).

Conclusions: The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

Show MeSH