The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

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.

Comparative proteomic analysis of Bactrocera dorsalis (Hendel) in response to thermal stress

“…In addition, two arginine kinases (3202 and 5204) were increased by low temperatures exposure in B. dorsalis. This enzyme catalyzes the conversion of ATP and L-arginine to ADP and N(omega)-phospho-Larginine, and so it regulates the cellular energy homeostasis during periods of high energy demand or energy supply fluctuations (Voncken et al, 2013). Hence our real-time PCR determinations confirmed higher levels also at RNA level.…”

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

“…First, we used available antibodies to known proteins such as two 14-3-3 proteins (60) that turned out to be enriched in the flagellar fraction. Because the repartition criterion is not definitive in the case of proteins present in both the cell body and the flagellum, we also investigated the arginine kinase, a protein known to exhibit such a profile (61) and that produced a relatively neutral score (1.27) but with a high number of peptides (supplemental Table S1). Second, in order to find new members present in the flagellum, we selected 10 proteins listed in supplemental Table S3 that were indicated as hypothetical for further biological validation.…”

“…AK is an enzyme of the phosphagen biosynthetic pathway and is encoded by three very closely related genes in T. brucei (62). The three encoded proteins share a highly conserved central domain with only tiny extensions providing protein specificity for cytoplasmic, glycosomal or flagellar localization (61). An anti-AK antiserum was raised in rabbits against the Trypanosoma cruzi protein where it stains the cytosol (62) but was reported to cross-react with its T. brucei counterparts by immunoblotting (63).…”

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

“…The second protein predicted to be myristoylated is the arginine kinase TbAK1 (Tb927.9.6170). TbAK1 is part of the phosphoarginine energy-buffering system and the C-terminally c-myc tagged protein localizes to the flagellum in both PCF and BSF trypanosomes (Voncken et al, 2013). However, biochemically the protein was not detected in the flagellar proteome (Oberholzer et al, 2011) but in mitochondrial membranes instead ).…”