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The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations - PubMed

  • ️Tue Jan 01 2013

The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations

Frank Voncken et al. PLoS One. 2013.

Abstract

Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Conserved sequence features of TbAK1-3.

Sequence alignment of TbAK1-3, T. cruzi TcCLB.507241.30, and representative arginine kinases from insects, crustaceans and molluscs. The N-terminal flagellar targeting signal of TbAK1 and the C-terminal PTS1 signal of TbAK2 are shown in bold face. Key residues involved in the binding of ATP and arginine are indicated on top of the alignment. The phosphagen kinase active site signature sequence is printed white bold face on a black background. The conserved α-helical N-terminal domain and C-terminal α/β saddle domain are boxed. Identical amino acids are represented by a dot and gaps are shown as dashes. See Material and Methods for different organism names and gene accession numbers.

Figure 2
Figure 2. TbAK1-nHis catalyses the reversible phosphorylation of arginine.

(A) TbAK1-nHis was expressed in E. coli BL21 (DE3) and subsequently isolated using Talon affinity chromatography. Left panel: SDS-PAGE and coomassie brilliant blue (CBB) staining revealed a single protein band (∼55 kDa) in the purified TbAK1-nHis protein fraction. Right panel: western blot (WB) analysis using a commercial His-tag antibody stained the same protein band (arrow). (B) HPLC analysis of the reaction products formed in the absence (left panel) or presence (right panel) of purified TbAK1-nHis. Elution time of arginine and phosphoarginine are 3.3 min and 5.5 min, respectively. (C) Reverse arginine kinase reaction catalysed by recombinant TbAK1-nHis and using ADP (1 mM) and increasing volumes (0–10 µl) of the collected phosphoarginine fraction as substrate. ATP formation from phospharginine was measured using a firefly luciferase-dependent ATP detection kit. Abbreviations are: M, molecular weight marker; kDa, kilodalton.

Figure 3
Figure 3. TbAK1-3 isoforms have different subcellular locations in T. brucei.

Immunofluorescence microscopy of PCF (A) and BSF (C) T. brucei 449 cell lines expressing different myc-tagged TbAK isoforms (red). Glycosomes (green) were detected using a polyclonal antibody directed against the glycosomal marker protein aldolase (Ald) , while the nucleus and kinetoplast were stained (blue) with DAPI. The merge (overlay) shows the co-localisation of TbArg2-nmyc and the glycosomal aldolase. Western blot analysis of PCF (B) and BSF (D) T. brucei 449 cell lines grown in the absence (–, non-induced) or presence (+, induced) of tetracycline (0.5 µg.mL−1), and using a commercial myc-antibody. For western blot analysis, cells were harvested 24 hours after induction and 2×106 trypanosomes were analysed per gel lane. Abbreviations: Phase, phase-contrast. (E, F) Immunofluorescence microscopy of PCF T. brucei 449 cell lines expressing GFP (E) and NtermTbAK1/GFP (F). GFP is visualized by its natural auto-fluorescence, while the nucleus and kinetoplast were stained (blue) with DAPI. Insets in E and F: western blot analysis using a commercial GFP-antibody confirmed the expression of GFP and the NtermTbAK1/GFP fusion protein.

Figure 4
Figure 4. Differential expression of TbAK1-3 in T. brucei.

(A) Northern blot analysis of total RNA (10 µg per lane) isolated from BSF and PCF T. brucei 449. The 3′UTRs of TbAK1-3 were used as DNA probes to detect the respective mRNA transcripts, while the signal recognition particle (SRP) was used as a loading control . Numbers represent the relative mRNA ratios (PCF/BSF) calculated from pixel intensities of the different mRNA bands (ImageJ:

http://rsb.info.nih.gov/ij/

) after normalisation against SRP, and represent the mean from 3 independent experiments. (B) Specific activities of arginine kinase in total cell lysates from the parental (no expression vector), control (containing pHU1 without insert), and myc-tagged TbAK1-3 expressing PCF (white bars) and BSF (black bars) T. brucei cell lines. Specific activity (µmol phosphoarginine formed per mg protein per min) was determined in the forward (phosphoarginine synthesis) direction using 2 mM arginine and 0.8 mM ATP as substrates, and using 10 µg of protein (total cell-lysate) per assay. Values are the means of at least three independent experiments.

Figure 5
Figure 5. Growth analysis of T. brucei 449 cell lines expressing myc-tagged TbAK1-3.

Growth curves of parental (PCF449: no vector), control (PCF449/pHU1: containing pHU1 without insert), and myc-tagged TbAK1-3 expressing PCF (A) and BSF (B) cell-lines under standard (solid lines) and hydrogen peroxide-challenged (dashed lines) culture conditions. Expression of myc-tagged TbAK1-3 was induced by addition of 0.5 µg.ml−1 tetracycline 24 hours before the start of the experiment. PCF trypanosomes were oxidatively challenged with 10 µM hydrogen peroxide, while BSF trypanosomes were challenged with 200 µM hydrogen peroxide. Cell densities were determined every 24 hours, for a total of 72 hours. Each growth curve represents the means of >3 independent experiments. Abbreviations: flag, flagellar; glyc, glycosomal; cyto, cytosolic.

Figure 6
Figure 6. Silencing of TbAK1-3 expression in PCF T. brucei.

(A) TbAK isoform-specific RT-PCR of the parental PCF EATRO1125.T7T cell line (lanes 2–4) and the derived TbAK-RNAi cell line (lanes 5–7) cultured for 6 days in the presence of tetracycline (0.5 µg.ml−1). No TbAK1 PCR-product was found in the absence (–) of reverse transcriptase (lane 1), indicating that the mRNA samples were not contaminated with genomic DNA. PCR of the non-related gene TbMCP11 (lane 8, [44]) was used as a positive control for cDNA synthesis. (B) Cumulative growth curves of the PCF EATRO1125.T7T cell line (solid line) and the tetracycline-induced TbAK-RNAi cell line (dashed line) showed an about 100-fold difference in cell density after 12 days under standard culture conditions. (C) Growth curves of the parental PCF EATRO1125.T7T cell line (solid lines) and the derived TbAK-RNAi cell line (dashed lines), grown under standard and oxidative challenging culture conditions. RNAi in the PCF TbAK-RNAi cell line was induced with tetracycline for 6 days prior to the start of the experiment, ensuring complete silencing of all TbAK isoforms (see (A)). For the oxidative-challenging culture conditions, 0.025 µM of hydrogen peroxide was added to the medium.

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References

    1. Ellington WR (2001) Evolution and physiological roles of phosphagen systems. Annu Rev Physiol 63: 289–325. - PubMed
    1. Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM (1992) Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit’ for cellular energy homeostasis. Biochem J 281 (Pt 1): 21–40. - PMC - PubMed
    1. Watts DC, Bannister LH (1970) Location of arginine kinase in the cilia of Tetrahymena pyriformis. Nature 226: 450–451. - PubMed
    1. Noguchi M, Sawada T, Akazawa T (2001) ATP-regenerating system in the cilia of Paramecium caudatum. J Exp Biol 204: 1063–1071. - PubMed
    1. Hoffman GG, Ellington WR (2011) Arginine kinase isoforms in the closest protozoan relative of metazoans. Comp Biochem Physiol Part D Genomics Proteomics 6: 171–177. - PubMed

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