Identification of a Bacterial-Like HslVU Protease in the Mitochondria of Trypanosoma brucei and Its Role in Mitochondrial DNA Replication

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.

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…Consistent with these data, we show that TbPOLID is highly stable (Fig. 2A), indicating that it is not regulated through proteolytic degradation by the mitochondrial protease HslVU (25). Although POLID levels remained essentially constant, we found a 4-fold increase in FI per TbPOLID focus and a corresponding decrease in FI within the mitochondrial matrix (equation 1).…”

“…Alternatively, changes in protein abundance also could account for this variation in POLID-PTP localization. The only mitochondrial protease that has been shown to regulate kDNA replication is TbHslVU (25). This bacterial-like protease regulates maxicircle replication through the degradation of the helicase TbPIF2 (27).…”

Structural and Biochemical Analyses of the Eukaryotic Heat Shock Locus V (HslV) from Trypanosoma brucei

“…Among them, the HslVU from the protozoan parasites, Leishmania, Trypanosoma, and Plasmodium, which cause significant human diseases, including African sleeping sickness, malaria, and leishmaniasis, have been experimentally characterized (26 -29). Intriguingly, HslU and HslV are localized in the mitochondria in Trypanosoma brucei, where they have a novel function in regulating mitochondrial DNA replication (28). The amino acid sequence of HslV from T. brucei (TbHslV) shares more than 40% identity with that of HslV from Escherichia coli (EcHslV; Fig.…”

“…There are two potential HslU molecules, HslU1 and HslU2, in T. brucei (TbHslU1 and TbHslU2) that have high identity with the HslU from E. coli (EcHslU; Fig. 1B) (28 In order to understand the molecular features of the eukaryotic HslVU system, we performed biochemical characterization of TbHslV and TbHslU by using a synthetic substrate, benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methyl coumarin (Z-GGL-AMC) (4,30) and a natural substrate of EcHslU, the SulA protein (31,32). Although both TbHslU1 and TbHslU2 regulate mitochondrial DNA replication (28), we found that only TbHslU2 acts as an activator of TbHslV protease.…”

“…The initial model was energy-minimized with SPBDV (45) and the CNS package (46). (28), which have N-terminal signal peptides that direct them to the mitochondria. Excluding the signal sequence, TbHslV shares 41.4% sequence identity with EcHslV and 46.3% sequence identity with HiHslV (Fig.…”