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.
Highlights
All eukaryotes contain energy buffering systems for the regulation of their energy homeostasis in periods of high-energy demand or energy supply fluctuations
The two most widely distributed energy buffering systems depend on the phosphorylation of creatine or arginine, with the phosphocreatine/creatine kinase system predominantly found in vertebrates and sponges, and the phosphoarginine/arginine kinase system mainly present in invertebrate organisms [1,2]
Several models have been postulated in which the spatial distribution of phosphagen kinases plays a crucial role in intracellular energy transport as well as in the control and regulation of ATP-dependent enzymes and pathways in the different subcellular compartments [2]
Summary
All eukaryotes contain energy buffering systems for the regulation of their energy homeostasis in periods of high-energy demand or energy supply fluctuations. The majority of these systems depend on phosphagen kinases that catalyse the reversible and ATP-dependent phosphorylation of guanidino acceptor compounds [1,2]. The derived high-energy phosphagens are relatively small and highly diffusible molecules that provide fast energy supply when energy consumption becomes critical [1] They stabilise the cellular ATP/ADP ratio and function as a temporal and spatial energy buffer in the cell [2]. Unicellular eukaryotes contain similar energy buffering systems, with phosphoarginine/arginine kinase systems described for the ciliates Paramecium caudatum and Tetrahymena pyriformis, the choanoflagellate Monosiga brevicollis, as well as for some Kinetoplastida [3,4,5,6,7,8]
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