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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 detection of minicircles and maxicircles in the kinetoplast by FISH.Cells were fixed, probed for minicircles (red) or maxicircles (green), and counterstained with DAPI. kDNA is indicated with a K and nucleus with an N. Bar: 2 µm. FISH does not detect covalently-closed DNA minicircles because they are non-denaturable [24]. This is likely also true for maxicircles. Thus the FISH signal may not be proportional to the total populations of minicircles and maxicircles. Bars: 2 µm.
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ppat-1000048-g007: In situ detection of minicircles and maxicircles in the kinetoplast by FISH.Cells were fixed, probed for minicircles (red) or maxicircles (green), and counterstained with DAPI. kDNA is indicated with a K and nucleus with an N. Bar: 2 µm. FISH does not detect covalently-closed DNA minicircles because they are non-denaturable [24]. This is likely also true for maxicircles. Thus the FISH signal may not be proportional to the total populations of minicircles and maxicircles. Bars: 2 µm.

Mentions: We used fluorescence in situ hybridization (FISH) to investigate the effect of TbHslV RNAi on distribution of minicircle and maxicircle DNAs within the network [24]. In control cells, the late-replicating network has the minicircles organized in a dumbbell shape with maxicircles clustered in the middle (Fig. 7a; see also [30], [35]–[37]). In an enlarged kinetoplast from a TbHslV RNAi cell, however, minicircle DNA was spread out over nearly the entire DAPI-stained network, whereas maxicircle DNA remained at the center (Fig. 7b). In abnormally segregating kinetoplasts, the minicircles also filled most of the DAPI-stained network, whereas the maxicircle was segregated into two symmetrical clusters (Fig. 7c). Due to the excessive size of minicircle network, it turned into a thread between the segregated maxicircles. Of 30 abnormally segregating kinetoplasts, 23 had segregated their maxicircles, in striking contrast to those in the control cells, which remained in the center (compare Figs. 7c and 7a). In the remaining 7, maxicircles have not segregated and appeared like the control (compare Figs. 7d and 7a). In the asymmetrically dividing kinetoplast, minicircle DNA was also distributed unevenly, constituting the basis of unevenly sized kinetoplasts, whereas maxicircle DNA was always symmetrically segregated (Fig. 7e). These results suggest that uneven segregation and enlargement of the kinetoplasts can be attributed to the excessive synthesis and uneven distribution of minicircles.


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 detection of minicircles and maxicircles in the kinetoplast by FISH.Cells were fixed, probed for minicircles (red) or maxicircles (green), and counterstained with DAPI. kDNA is indicated with a K and nucleus with an N. Bar: 2 µm. FISH does not detect covalently-closed DNA minicircles because they are non-denaturable [24]. This is likely also true for maxicircles. Thus the FISH signal may not be proportional to the total populations of minicircles and maxicircles. Bars: 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000048-g007: In situ detection of minicircles and maxicircles in the kinetoplast by FISH.Cells were fixed, probed for minicircles (red) or maxicircles (green), and counterstained with DAPI. kDNA is indicated with a K and nucleus with an N. Bar: 2 µm. FISH does not detect covalently-closed DNA minicircles because they are non-denaturable [24]. This is likely also true for maxicircles. Thus the FISH signal may not be proportional to the total populations of minicircles and maxicircles. Bars: 2 µm.
Mentions: We used fluorescence in situ hybridization (FISH) to investigate the effect of TbHslV RNAi on distribution of minicircle and maxicircle DNAs within the network [24]. In control cells, the late-replicating network has the minicircles organized in a dumbbell shape with maxicircles clustered in the middle (Fig. 7a; see also [30], [35]–[37]). In an enlarged kinetoplast from a TbHslV RNAi cell, however, minicircle DNA was spread out over nearly the entire DAPI-stained network, whereas maxicircle DNA remained at the center (Fig. 7b). In abnormally segregating kinetoplasts, the minicircles also filled most of the DAPI-stained network, whereas the maxicircle was segregated into two symmetrical clusters (Fig. 7c). Due to the excessive size of minicircle network, it turned into a thread between the segregated maxicircles. Of 30 abnormally segregating kinetoplasts, 23 had segregated their maxicircles, in striking contrast to those in the control cells, which remained in the center (compare Figs. 7c and 7a). In the remaining 7, maxicircles have not segregated and appeared like the control (compare Figs. 7d and 7a). In the asymmetrically dividing kinetoplast, minicircle DNA was also distributed unevenly, constituting the basis of unevenly sized kinetoplasts, whereas maxicircle DNA was always symmetrically segregated (Fig. 7e). These results suggest that uneven segregation and enlargement of the kinetoplasts can be attributed to the excessive synthesis and uneven distribution of minicircles.

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