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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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Effect of TbHslU1+2 double RNAi on free minicircle replication intermediates.(A). Total DNA was fractionated on an agarose/ethidium gel and a Southern blot was probed for minicircles. N/G, nicked/gapped minicircles; L, linearized minicircles; CC, covalently-closed minicircles, *, nonspecific hybridization to nuclear DNA. The nicked/gapped minicircles form a doublet with the lower component possibly linearized minicircles. Since it is present prior to RNAi induction (day 0), it is likely unrelated to RNAi. (B). Quantitation (by Phosphorimager) of bands from A. The nicked/gapped species includes both components of the doublet.
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ppat-1000048-g005: Effect of TbHslU1+2 double RNAi on free minicircle replication intermediates.(A). Total DNA was fractionated on an agarose/ethidium gel and a Southern blot was probed for minicircles. N/G, nicked/gapped minicircles; L, linearized minicircles; CC, covalently-closed minicircles, *, nonspecific hybridization to nuclear DNA. The nicked/gapped minicircles form a doublet with the lower component possibly linearized minicircles. Since it is present prior to RNAi induction (day 0), it is likely unrelated to RNAi. (B). Quantitation (by Phosphorimager) of bands from A. The nicked/gapped species includes both components of the doublet.

Mentions: To analyze the effect of TbHslVU RNAi on the free minicircle species, we fractionated total DNA from control and TbHslU1+2 RNAi cells on an agarose gel in the presence of ethidium bromide to resolve covalently-closed free minicircles from those containing gaps [29]. Probing a Southern blot for minicircles revealed that the levels of covalently-closed and gapped free minicircles remained constant during the first 5 days of RNAi and then dramatically increased by 5 to 6-fold by the end of the 9 day experiment (Fig. 5), implying that silencing of TbHslU enhances the rate of minicircle replication.


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)

Effect of TbHslU1+2 double RNAi on free minicircle replication intermediates.(A). Total DNA was fractionated on an agarose/ethidium gel and a Southern blot was probed for minicircles. N/G, nicked/gapped minicircles; L, linearized minicircles; CC, covalently-closed minicircles, *, nonspecific hybridization to nuclear DNA. The nicked/gapped minicircles form a doublet with the lower component possibly linearized minicircles. Since it is present prior to RNAi induction (day 0), it is likely unrelated to RNAi. (B). Quantitation (by Phosphorimager) of bands from A. The nicked/gapped species includes both components of the doublet.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000048-g005: Effect of TbHslU1+2 double RNAi on free minicircle replication intermediates.(A). Total DNA was fractionated on an agarose/ethidium gel and a Southern blot was probed for minicircles. N/G, nicked/gapped minicircles; L, linearized minicircles; CC, covalently-closed minicircles, *, nonspecific hybridization to nuclear DNA. The nicked/gapped minicircles form a doublet with the lower component possibly linearized minicircles. Since it is present prior to RNAi induction (day 0), it is likely unrelated to RNAi. (B). Quantitation (by Phosphorimager) of bands from A. The nicked/gapped species includes both components of the doublet.
Mentions: To analyze the effect of TbHslVU RNAi on the free minicircle species, we fractionated total DNA from control and TbHslU1+2 RNAi cells on an agarose gel in the presence of ethidium bromide to resolve covalently-closed free minicircles from those containing gaps [29]. Probing a Southern blot for minicircles revealed that the levels of covalently-closed and gapped free minicircles remained constant during the first 5 days of RNAi and then dramatically increased by 5 to 6-fold by the end of the 9 day experiment (Fig. 5), implying that silencing of TbHslU enhances the rate of minicircle replication.

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