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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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Enzymatic activity and intracellular localization of TbHslVU.(A). TbHslV contains the conserved threonine and lysine residues (arrows) found essential for the activities of HslV in E. coli [6] and the β-subunits of 20S CP in T. brucei [54]. (B). The ATP-dependent peptidase activity of TbHslV. Wild type and three TbHslV mutants T20A, T21A and K53A were expressed as HA-tagged proteins in T. brucei, immunoprecipitated and assayed for hydrolysis of Cbz-Gly-Gly-Leu-AMC. (C). Cells stably expressing TbHslVU-HA were labeled for mitochondria with Mitotracker green dye (green), immunostained with anti-HA mAb for HA-tagged proteins (red) and counterstained with DAPI for DNA (blue). Arrows indicate the focal points of HA-staining corresponding to the positions of kinetoplasts (arrowheads). (D). Cells expressing TbHslVU-HA with the putative mitochondrial targeting sequences deleted were stained with anti-HA antibody (red) and counterstained with DAPI. Bars: 2 µm.
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ppat-1000048-g001: Enzymatic activity and intracellular localization of TbHslVU.(A). TbHslV contains the conserved threonine and lysine residues (arrows) found essential for the activities of HslV in E. coli [6] and the β-subunits of 20S CP in T. brucei [54]. (B). The ATP-dependent peptidase activity of TbHslV. Wild type and three TbHslV mutants T20A, T21A and K53A were expressed as HA-tagged proteins in T. brucei, immunoprecipitated and assayed for hydrolysis of Cbz-Gly-Gly-Leu-AMC. (C). Cells stably expressing TbHslVU-HA were labeled for mitochondria with Mitotracker green dye (green), immunostained with anti-HA mAb for HA-tagged proteins (red) and counterstained with DAPI for DNA (blue). Arrows indicate the focal points of HA-staining corresponding to the positions of kinetoplasts (arrowheads). (D). Cells expressing TbHslVU-HA with the putative mitochondrial targeting sequences deleted were stained with anti-HA antibody (red) and counterstained with DAPI. Bars: 2 µm.

Mentions: We identified in the trypanosome genome database (www.genedb.org) an HslV homolog (Tb11.01.2000; designated TbHslV) with ∼40% identity to bacterial HslV (Fig. S1A) and a 15–24% overall identity to the three catalytic β-subunits in T. brucei 20S CP (data not shown). In addition, we found two HslU homologs, TbHslU1 (Tb927.5.1520) and TbHslU2 (Tb11.01.4050), that are 40–44% identical to E. coli HslU and ∼40% identical to each other (Fig. S2A). These proteins have potential N-terminal mitochondrial targeting signals. In addition, TbHslV has two threonines (T20 and T21) next to the targeting signal and a downstream lysine at position 53 (Fig. 1A, arrows). Both TbHslU1 and TbHslU2 possess the putative NTP-binding motif (P-loop) and the conserved residues essential for the ATPase activity of HslU (Fig. S2A, arrows). By homology modeling [31], TbHslV, TbHslU1 and TbHslU2 can be folded into three-dimensional structures resembling those of the HslV and HslU of E. coli (Figs. S1B and S2B; [4].


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)

Enzymatic activity and intracellular localization of TbHslVU.(A). TbHslV contains the conserved threonine and lysine residues (arrows) found essential for the activities of HslV in E. coli [6] and the β-subunits of 20S CP in T. brucei [54]. (B). The ATP-dependent peptidase activity of TbHslV. Wild type and three TbHslV mutants T20A, T21A and K53A were expressed as HA-tagged proteins in T. brucei, immunoprecipitated and assayed for hydrolysis of Cbz-Gly-Gly-Leu-AMC. (C). Cells stably expressing TbHslVU-HA were labeled for mitochondria with Mitotracker green dye (green), immunostained with anti-HA mAb for HA-tagged proteins (red) and counterstained with DAPI for DNA (blue). Arrows indicate the focal points of HA-staining corresponding to the positions of kinetoplasts (arrowheads). (D). Cells expressing TbHslVU-HA with the putative mitochondrial targeting sequences deleted were stained with anti-HA antibody (red) and counterstained with DAPI. Bars: 2 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2277460&req=5

ppat-1000048-g001: Enzymatic activity and intracellular localization of TbHslVU.(A). TbHslV contains the conserved threonine and lysine residues (arrows) found essential for the activities of HslV in E. coli [6] and the β-subunits of 20S CP in T. brucei [54]. (B). The ATP-dependent peptidase activity of TbHslV. Wild type and three TbHslV mutants T20A, T21A and K53A were expressed as HA-tagged proteins in T. brucei, immunoprecipitated and assayed for hydrolysis of Cbz-Gly-Gly-Leu-AMC. (C). Cells stably expressing TbHslVU-HA were labeled for mitochondria with Mitotracker green dye (green), immunostained with anti-HA mAb for HA-tagged proteins (red) and counterstained with DAPI for DNA (blue). Arrows indicate the focal points of HA-staining corresponding to the positions of kinetoplasts (arrowheads). (D). Cells expressing TbHslVU-HA with the putative mitochondrial targeting sequences deleted were stained with anti-HA antibody (red) and counterstained with DAPI. Bars: 2 µm.
Mentions: We identified in the trypanosome genome database (www.genedb.org) an HslV homolog (Tb11.01.2000; designated TbHslV) with ∼40% identity to bacterial HslV (Fig. S1A) and a 15–24% overall identity to the three catalytic β-subunits in T. brucei 20S CP (data not shown). In addition, we found two HslU homologs, TbHslU1 (Tb927.5.1520) and TbHslU2 (Tb11.01.4050), that are 40–44% identical to E. coli HslU and ∼40% identical to each other (Fig. S2A). These proteins have potential N-terminal mitochondrial targeting signals. In addition, TbHslV has two threonines (T20 and T21) next to the targeting signal and a downstream lysine at position 53 (Fig. 1A, arrows). Both TbHslU1 and TbHslU2 possess the putative NTP-binding motif (P-loop) and the conserved residues essential for the ATPase activity of HslU (Fig. S2A, arrows). By homology modeling [31], TbHslV, TbHslU1 and TbHslU2 can be folded into three-dimensional structures resembling those of the HslV and HslU of E. coli (Figs. S1B and S2B; [4].

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