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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.

Show MeSH
A cartoon illustrating the components studied here (drawn roughly to scale) that lie within the sphere of influence of mtDNA. (A) A cluster of approximately eight mitochondrial genomes (see Table 1; only three are shown) are tethered through the mitochondrial membranes (see Figure 2) to kinesin and cytoplasmic microtubular network (see Figure 3); tethering restricts motion (see Figure 4), but the molecular mechanism is unknown. A cluster is usually found in a thin part of the mitochondrion poor in YFP-cytochrome oxidase (Figure 1G), and mitochondria generally split near Drp1 foci lying ~300 nm away (Figure 5). (B) A high-power view of a region in (A). Nascent mtRNA is translated cotranscriptionally by mitochondrial ribosomes bound both to the polymerase [40] and the inner mitochondrial membrane [57]; completed mRNAs (not shown) are also translated during most of their lifetime in this region (Figure 7J). The cytoplasmic translation machinery that makes nuclear-encoded proteins destined for the mitochondrion – marked by a ribosomal protein (S6) and a chaperone (NAC) – lie immediately on the other side of the mitochondrial membrane (Figure 8A,8B,8C,8D,8E,8F,8G,8H). Here, a cytoplasmic peptide is being made and imported through the translocases in the outer and inner membranes (TOM and TIM; TOM is marked by Tom22; Figure 8I,8J,8K,8L) where it will assemble with a mitochondrial-encoded peptide in the inner mitochondrial membrane. The close proximity of the two sets of machinery on each side of the membranes ensures efficient assembly of mitochondrial complexes containing proteins encoded by nuclear and mitochondrial genomes.
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Figure 9: A cartoon illustrating the components studied here (drawn roughly to scale) that lie within the sphere of influence of mtDNA. (A) A cluster of approximately eight mitochondrial genomes (see Table 1; only three are shown) are tethered through the mitochondrial membranes (see Figure 2) to kinesin and cytoplasmic microtubular network (see Figure 3); tethering restricts motion (see Figure 4), but the molecular mechanism is unknown. A cluster is usually found in a thin part of the mitochondrion poor in YFP-cytochrome oxidase (Figure 1G), and mitochondria generally split near Drp1 foci lying ~300 nm away (Figure 5). (B) A high-power view of a region in (A). Nascent mtRNA is translated cotranscriptionally by mitochondrial ribosomes bound both to the polymerase [40] and the inner mitochondrial membrane [57]; completed mRNAs (not shown) are also translated during most of their lifetime in this region (Figure 7J). The cytoplasmic translation machinery that makes nuclear-encoded proteins destined for the mitochondrion – marked by a ribosomal protein (S6) and a chaperone (NAC) – lie immediately on the other side of the mitochondrial membrane (Figure 8A,8B,8C,8D,8E,8F,8G,8H). Here, a cytoplasmic peptide is being made and imported through the translocases in the outer and inner membranes (TOM and TIM; TOM is marked by Tom22; Figure 8I,8J,8K,8L) where it will assemble with a mitochondrial-encoded peptide in the inner mitochondrial membrane. The close proximity of the two sets of machinery on each side of the membranes ensures efficient assembly of mitochondrial complexes containing proteins encoded by nuclear and mitochondrial genomes.

Mentions: The position of mitochondria within the cell is determined largely by the cytoskeleton. For example, the yeast nucleoid is anchored to the cytoskeleton through the inner mitochondrial membrane via Mgm1p (in the inter-membrane space) and Mmm1p (in the outer membrane) [2,7,8,48,49]; this anchorage provides a mechanism whereby mtDNA might be replicated, segregated, and inherited [46]. Our results suggest that many of the clusters of 6–10 mitochondrial genomes in a human cell have an even more extensive sphere of influence that includes the translation machineries on both sides of the mitochondrial membranes (Figure 9). 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. Moreover, depleting mitochondrial DNA should directly affect mitochondrial structure, which it does [50]. According to this view, mtDNA acts as a central hub that organizes its immediate surroundings both within – and outside – the mitochondrion; one result would be an integration of mitochondrial and cellular protein synthesis.


The functional organization of mitochondrial genomes in human cells.

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

A cartoon illustrating the components studied here (drawn roughly to scale) that lie within the sphere of influence of mtDNA. (A) A cluster of approximately eight mitochondrial genomes (see Table 1; only three are shown) are tethered through the mitochondrial membranes (see Figure 2) to kinesin and cytoplasmic microtubular network (see Figure 3); tethering restricts motion (see Figure 4), but the molecular mechanism is unknown. A cluster is usually found in a thin part of the mitochondrion poor in YFP-cytochrome oxidase (Figure 1G), and mitochondria generally split near Drp1 foci lying ~300 nm away (Figure 5). (B) A high-power view of a region in (A). Nascent mtRNA is translated cotranscriptionally by mitochondrial ribosomes bound both to the polymerase [40] and the inner mitochondrial membrane [57]; completed mRNAs (not shown) are also translated during most of their lifetime in this region (Figure 7J). The cytoplasmic translation machinery that makes nuclear-encoded proteins destined for the mitochondrion – marked by a ribosomal protein (S6) and a chaperone (NAC) – lie immediately on the other side of the mitochondrial membrane (Figure 8A,8B,8C,8D,8E,8F,8G,8H). Here, a cytoplasmic peptide is being made and imported through the translocases in the outer and inner membranes (TOM and TIM; TOM is marked by Tom22; Figure 8I,8J,8K,8L) where it will assemble with a mitochondrial-encoded peptide in the inner mitochondrial membrane. The close proximity of the two sets of machinery on each side of the membranes ensures efficient assembly of mitochondrial complexes containing proteins encoded by nuclear and mitochondrial genomes.
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Related In: Results  -  Collection

Show All Figures
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Figure 9: A cartoon illustrating the components studied here (drawn roughly to scale) that lie within the sphere of influence of mtDNA. (A) A cluster of approximately eight mitochondrial genomes (see Table 1; only three are shown) are tethered through the mitochondrial membranes (see Figure 2) to kinesin and cytoplasmic microtubular network (see Figure 3); tethering restricts motion (see Figure 4), but the molecular mechanism is unknown. A cluster is usually found in a thin part of the mitochondrion poor in YFP-cytochrome oxidase (Figure 1G), and mitochondria generally split near Drp1 foci lying ~300 nm away (Figure 5). (B) A high-power view of a region in (A). Nascent mtRNA is translated cotranscriptionally by mitochondrial ribosomes bound both to the polymerase [40] and the inner mitochondrial membrane [57]; completed mRNAs (not shown) are also translated during most of their lifetime in this region (Figure 7J). The cytoplasmic translation machinery that makes nuclear-encoded proteins destined for the mitochondrion – marked by a ribosomal protein (S6) and a chaperone (NAC) – lie immediately on the other side of the mitochondrial membrane (Figure 8A,8B,8C,8D,8E,8F,8G,8H). Here, a cytoplasmic peptide is being made and imported through the translocases in the outer and inner membranes (TOM and TIM; TOM is marked by Tom22; Figure 8I,8J,8K,8L) where it will assemble with a mitochondrial-encoded peptide in the inner mitochondrial membrane. The close proximity of the two sets of machinery on each side of the membranes ensures efficient assembly of mitochondrial complexes containing proteins encoded by nuclear and mitochondrial genomes.
Mentions: The position of mitochondria within the cell is determined largely by the cytoskeleton. For example, the yeast nucleoid is anchored to the cytoskeleton through the inner mitochondrial membrane via Mgm1p (in the inter-membrane space) and Mmm1p (in the outer membrane) [2,7,8,48,49]; this anchorage provides a mechanism whereby mtDNA might be replicated, segregated, and inherited [46]. Our results suggest that many of the clusters of 6–10 mitochondrial genomes in a human cell have an even more extensive sphere of influence that includes the translation machineries on both sides of the mitochondrial membranes (Figure 9). 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. Moreover, depleting mitochondrial DNA should directly affect mitochondrial structure, which it does [50]. According to this view, mtDNA acts as a central hub that organizes its immediate surroundings both within – and outside – the mitochondrion; one result would be an integration of mitochondrial and cellular protein synthesis.

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