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Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

Li Z, Lindsay ME, Motyka SA, Englund PT, Wang CC - PLoS Pathog. (2008)

Bottom Line: By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA).TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote.It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, United States of America.

ABSTRACT
ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

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Electron microscopic examination of kDNA networks from the control and TbHslV RNAi cells.Methods were described by [38]. (A, B, E, F) kDNA networks from cells after 7 days of TbHslV RNAi. (H) A kDNA network from an un-induced control cell. (C, D) Enlargements of the network in A, corresponding to the areas outlined in white framed boxes. (G) Enlargement of the network in F framed in a white box. The arrow in G indicates a maxicircle. Scale bars for A, B, E, F, and H, 2 µm and for C, D, and G, 0.5 µm.
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ppat-1000048-g004: Electron microscopic examination of kDNA networks from the control and TbHslV RNAi cells.Methods were described by [38]. (A, B, E, F) kDNA networks from cells after 7 days of TbHslV RNAi. (H) A kDNA network from an un-induced control cell. (C, D) Enlargements of the network in A, corresponding to the areas outlined in white framed boxes. (G) Enlargement of the network in F framed in a white box. The arrow in G indicates a maxicircle. Scale bars for A, B, E, F, and H, 2 µm and for C, D, and G, 0.5 µm.

Mentions: This conclusion was confirmed by EM of isolated networks. Fig. 4H shows a network from a control cell with a typical elliptical shape and planar structure; it is about ∼6 µm in length and ∼3 µm in width, a standard size of kinetoplast after being processed for electron microscopy. Those from cells after 7 days of TbHslV RNAi were grossly enlarged, heterogeneous in size and irregular in shape with estimated lengths ranging from ∼10 µm to ∼16 µm (Figs. 4A, 4B, 4E and 4F). Electron-dense fibers were present in these enlarged networks. The one in Fig. 4F, apparently undergoing asymmetrical division, has, like the wild type, a cluster of maxicircles located between the two lobes (Fig. 4G).


Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

Li Z, Lindsay ME, Motyka SA, Englund PT, Wang CC - PLoS Pathog. (2008)

Electron microscopic examination of kDNA networks from the control and TbHslV RNAi cells.Methods were described by [38]. (A, B, E, F) kDNA networks from cells after 7 days of TbHslV RNAi. (H) A kDNA network from an un-induced control cell. (C, D) Enlargements of the network in A, corresponding to the areas outlined in white framed boxes. (G) Enlargement of the network in F framed in a white box. The arrow in G indicates a maxicircle. Scale bars for A, B, E, F, and H, 2 µm and for C, D, and G, 0.5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2277460&req=5

ppat-1000048-g004: Electron microscopic examination of kDNA networks from the control and TbHslV RNAi cells.Methods were described by [38]. (A, B, E, F) kDNA networks from cells after 7 days of TbHslV RNAi. (H) A kDNA network from an un-induced control cell. (C, D) Enlargements of the network in A, corresponding to the areas outlined in white framed boxes. (G) Enlargement of the network in F framed in a white box. The arrow in G indicates a maxicircle. Scale bars for A, B, E, F, and H, 2 µm and for C, D, and G, 0.5 µm.
Mentions: This conclusion was confirmed by EM of isolated networks. Fig. 4H shows a network from a control cell with a typical elliptical shape and planar structure; it is about ∼6 µm in length and ∼3 µm in width, a standard size of kinetoplast after being processed for electron microscopy. Those from cells after 7 days of TbHslV RNAi were grossly enlarged, heterogeneous in size and irregular in shape with estimated lengths ranging from ∼10 µm to ∼16 µm (Figs. 4A, 4B, 4E and 4F). Electron-dense fibers were present in these enlarged networks. The one in Fig. 4F, apparently undergoing asymmetrical division, has, like the wild type, a cluster of maxicircles located between the two lobes (Fig. 4G).

Bottom Line: By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA).TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote.It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, United States of America.

ABSTRACT
ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

Show MeSH
Related in: MedlinePlus