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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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TbHslVU RNAi led to heterogeneously sized kinetoplasts.(A). Flow cytometry analysis of DHE stained cells. A total of 25,000 cells were counted in each experiment (Left panel). DHE stains exclusively the kinetoplasts in control and RNAi cells (Right panel). (B). Southern analysis of changes in minicircle and maxicircle DNA content during TbHslU RNAi. The kinetics of minicircle (open circle) and maxicircle (filled square) accumulation are presented to the right of the Southern blots. (C). DAPI staining of the isolated kDNA networks. (D). Surface areas of the isolated kDNA networks stained with DAPI, and measured with the NIH Image software.
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ppat-1000048-g003: TbHslVU RNAi led to heterogeneously sized kinetoplasts.(A). Flow cytometry analysis of DHE stained cells. A total of 25,000 cells were counted in each experiment (Left panel). DHE stains exclusively the kinetoplasts in control and RNAi cells (Right panel). (B). Southern analysis of changes in minicircle and maxicircle DNA content during TbHslU RNAi. The kinetics of minicircle (open circle) and maxicircle (filled square) accumulation are presented to the right of the Southern blots. (C). DAPI staining of the isolated kDNA networks. (D). Surface areas of the isolated kDNA networks stained with DAPI, and measured with the NIH Image software.

Mentions: As another approach to assess kinetoplast size, we used dihydroethidium (DHE) that selectively stains the kDNA but not the nuclear DNA (Fig. 3A, Right panel) (DHE is oxidized to ethidium in the mitochondrion but not in the nucleus, thus staining only kDNA [32]). By flow cytometry, the DHE-stained TbHslV and TbHslU1+2 knockdown cells (7 days after RNAi) had a much broader distribution of fluorescence with higher intensity than that of the control cells (Fig. 3A, Left panel), indicating that the average kDNA/cell increases following RNAi.


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)

TbHslVU RNAi led to heterogeneously sized kinetoplasts.(A). Flow cytometry analysis of DHE stained cells. A total of 25,000 cells were counted in each experiment (Left panel). DHE stains exclusively the kinetoplasts in control and RNAi cells (Right panel). (B). Southern analysis of changes in minicircle and maxicircle DNA content during TbHslU RNAi. The kinetics of minicircle (open circle) and maxicircle (filled square) accumulation are presented to the right of the Southern blots. (C). DAPI staining of the isolated kDNA networks. (D). Surface areas of the isolated kDNA networks stained with DAPI, and measured with the NIH Image software.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000048-g003: TbHslVU RNAi led to heterogeneously sized kinetoplasts.(A). Flow cytometry analysis of DHE stained cells. A total of 25,000 cells were counted in each experiment (Left panel). DHE stains exclusively the kinetoplasts in control and RNAi cells (Right panel). (B). Southern analysis of changes in minicircle and maxicircle DNA content during TbHslU RNAi. The kinetics of minicircle (open circle) and maxicircle (filled square) accumulation are presented to the right of the Southern blots. (C). DAPI staining of the isolated kDNA networks. (D). Surface areas of the isolated kDNA networks stained with DAPI, and measured with the NIH Image software.
Mentions: As another approach to assess kinetoplast size, we used dihydroethidium (DHE) that selectively stains the kDNA but not the nuclear DNA (Fig. 3A, Right panel) (DHE is oxidized to ethidium in the mitochondrion but not in the nucleus, thus staining only kDNA [32]). By flow cytometry, the DHE-stained TbHslV and TbHslU1+2 knockdown cells (7 days after RNAi) had a much broader distribution of fluorescence with higher intensity than that of the control cells (Fig. 3A, Left panel), indicating that the average kDNA/cell increases following RNAi.

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