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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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In situ TdT-catalyzed Fluorescein-dUTP labeling in cells after 7 days of TbHslV RNAi.(A). The control cells (a–f). (B, C). The TbHslV RNAi cells (g–p). Percentages of TdT-labeled cells in control and TbHslV RNAi cells are presented (k). Bars: 2 µm.
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ppat-1000048-g006: In situ TdT-catalyzed Fluorescein-dUTP labeling in cells after 7 days of TbHslV RNAi.(A). The control cells (a–f). (B, C). The TbHslV RNAi cells (g–p). Percentages of TdT-labeled cells in control and TbHslV RNAi cells are presented (k). Bars: 2 µm.

Mentions: To investigate the organization of the replicating kinetoplast in RNAi cells, we in situ labeled gapped minicircles (and maxicircles) at 3′-OH groups using terminal deoxynucleotidyl transferase (TdT) and fluorescent deoxyuridine triphosphate [33],[34]. In control cells, we detected no TdT labeling of kDNA before kinetoplast replication as all minicircles are covalently closed (Fig. 6A, a). At the early stage of kinetoplast replication, there is strong TdT labeling at the two antipodal sites enriched in multiply-gapped free minicircles, not yet attached to the network (Fig. 6A, b). At the late stage of replication, when many gapped minicircles had attached to the network, TdT-label is still strong in the antipodal sites, but the network, especially the polar regions, are also labeled weakly because they contain minicircles which had most but not all of their gaps repaired just prior to network attachment (Fig. 6A, c). When the kinetoplast was undergoing segregation, TdT label spread over the entire network (Fig. 6A, d) until the completion of segregation when all the minicircles became covalently closed and could no longer be labeled (Fig. 6A, e–f).


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)

In situ TdT-catalyzed Fluorescein-dUTP labeling in cells after 7 days of TbHslV RNAi.(A). The control cells (a–f). (B, C). The TbHslV RNAi cells (g–p). Percentages of TdT-labeled cells in control and TbHslV RNAi cells are presented (k). Bars: 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000048-g006: In situ TdT-catalyzed Fluorescein-dUTP labeling in cells after 7 days of TbHslV RNAi.(A). The control cells (a–f). (B, C). The TbHslV RNAi cells (g–p). Percentages of TdT-labeled cells in control and TbHslV RNAi cells are presented (k). Bars: 2 µm.
Mentions: To investigate the organization of the replicating kinetoplast in RNAi cells, we in situ labeled gapped minicircles (and maxicircles) at 3′-OH groups using terminal deoxynucleotidyl transferase (TdT) and fluorescent deoxyuridine triphosphate [33],[34]. In control cells, we detected no TdT labeling of kDNA before kinetoplast replication as all minicircles are covalently closed (Fig. 6A, a). At the early stage of kinetoplast replication, there is strong TdT labeling at the two antipodal sites enriched in multiply-gapped free minicircles, not yet attached to the network (Fig. 6A, b). At the late stage of replication, when many gapped minicircles had attached to the network, TdT-label is still strong in the antipodal sites, but the network, especially the polar regions, are also labeled weakly because they contain minicircles which had most but not all of their gaps repaired just prior to network attachment (Fig. 6A, c). When the kinetoplast was undergoing segregation, TdT label spread over the entire network (Fig. 6A, d) until the completion of segregation when all the minicircles became covalently closed and could no longer be labeled (Fig. 6A, e–f).

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