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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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RNAi knockdown of TbHslVU affects cell growth and kinetoplast morphology.(A). Cells were grown in the presence (+) of tetracycline to induce RNAi for 7 days, and cell growth was monitored daily. Northern blots were performed to assess levels of TbHslV, TbHslU1 and TbHslU2 mRNA before (−) and after (+) 2 days of RNAi (insets). (B–E). Un-induced control cells (B) and cells after RNAi induction for 7 days (C) were labeled with YL1/2 antibody for basal bodies (BB, arrowheads) and counterstained with DAPI for nucleus (N) and kinetoplast (arrows). (D). Two TbHslVU RNAi cells at the final stage of cell division were still connected by a thin thread of kinetoplast DNA (arrows) between two basal bodies (arrowheads) in two well-separated cells. Bars: 2 µm. (E). Tabulation of RNAi cells with kinetoplasts in varying sizes and morphologies. Approximately 200 cells were counted at each time point and the data represent averages from three independent experiments.
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ppat-1000048-g002: RNAi knockdown of TbHslVU affects cell growth and kinetoplast morphology.(A). Cells were grown in the presence (+) of tetracycline to induce RNAi for 7 days, and cell growth was monitored daily. Northern blots were performed to assess levels of TbHslV, TbHslU1 and TbHslU2 mRNA before (−) and after (+) 2 days of RNAi (insets). (B–E). Un-induced control cells (B) and cells after RNAi induction for 7 days (C) were labeled with YL1/2 antibody for basal bodies (BB, arrowheads) and counterstained with DAPI for nucleus (N) and kinetoplast (arrows). (D). Two TbHslVU RNAi cells at the final stage of cell division were still connected by a thin thread of kinetoplast DNA (arrows) between two basal bodies (arrowheads) in two well-separated cells. Bars: 2 µm. (E). Tabulation of RNAi cells with kinetoplasts in varying sizes and morphologies. Approximately 200 cells were counted at each time point and the data represent averages from three independent experiments.

Mentions: To evaluate the function of TbHslVU, we used RNAi to knock down expression of each of the three subunits in procyclic trypanosomes. Knockdown of individual transcripts, confirmed by Northern blots (Fig. 2A, insets), resulted in modest to strong growth inhibition. Knockdown of TbHslV registered the highest inhibitory effect (Fig. 2A). Simultaneous knockdown of TbHslU1 and TbHslU2 led to a larger growth defect than that from individual knockdowns, though still not as severe as that from a TbHslV knockdown (Fig. 2A). DAPI staining showed significant changes in the size and shape of kinetoplasts in the RNAi cells (Fig. 2C), suggesting that TbHslVU could be involved in controlling replication and/or segregation of the kDNA network.


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)

RNAi knockdown of TbHslVU affects cell growth and kinetoplast morphology.(A). Cells were grown in the presence (+) of tetracycline to induce RNAi for 7 days, and cell growth was monitored daily. Northern blots were performed to assess levels of TbHslV, TbHslU1 and TbHslU2 mRNA before (−) and after (+) 2 days of RNAi (insets). (B–E). Un-induced control cells (B) and cells after RNAi induction for 7 days (C) were labeled with YL1/2 antibody for basal bodies (BB, arrowheads) and counterstained with DAPI for nucleus (N) and kinetoplast (arrows). (D). Two TbHslVU RNAi cells at the final stage of cell division were still connected by a thin thread of kinetoplast DNA (arrows) between two basal bodies (arrowheads) in two well-separated cells. Bars: 2 µm. (E). Tabulation of RNAi cells with kinetoplasts in varying sizes and morphologies. Approximately 200 cells were counted at each time point and the data represent averages from three independent experiments.
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Related In: Results  -  Collection

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

ppat-1000048-g002: RNAi knockdown of TbHslVU affects cell growth and kinetoplast morphology.(A). Cells were grown in the presence (+) of tetracycline to induce RNAi for 7 days, and cell growth was monitored daily. Northern blots were performed to assess levels of TbHslV, TbHslU1 and TbHslU2 mRNA before (−) and after (+) 2 days of RNAi (insets). (B–E). Un-induced control cells (B) and cells after RNAi induction for 7 days (C) were labeled with YL1/2 antibody for basal bodies (BB, arrowheads) and counterstained with DAPI for nucleus (N) and kinetoplast (arrows). (D). Two TbHslVU RNAi cells at the final stage of cell division were still connected by a thin thread of kinetoplast DNA (arrows) between two basal bodies (arrowheads) in two well-separated cells. Bars: 2 µm. (E). Tabulation of RNAi cells with kinetoplasts in varying sizes and morphologies. Approximately 200 cells were counted at each time point and the data represent averages from three independent experiments.
Mentions: To evaluate the function of TbHslVU, we used RNAi to knock down expression of each of the three subunits in procyclic trypanosomes. Knockdown of individual transcripts, confirmed by Northern blots (Fig. 2A, insets), resulted in modest to strong growth inhibition. Knockdown of TbHslV registered the highest inhibitory effect (Fig. 2A). Simultaneous knockdown of TbHslU1 and TbHslU2 led to a larger growth defect than that from individual knockdowns, though still not as severe as that from a TbHslV knockdown (Fig. 2A). DAPI staining showed significant changes in the size and shape of kinetoplasts in the RNAi cells (Fig. 2C), suggesting that TbHslVU could be involved in controlling replication and/or segregation of the kDNA network.

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