The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

Abstract

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.

Both sequence and context are important for flagellar targeting of a glucose transporter

“…This observation is not surprising, since most of these proteins are from mammals that are highly divergent from ancient parasitic protozoa. In addition, this sequence is not related to those involved in flagellar localization of non-membrane proteins that have been identified among the kinetoplastid protozoa (Luginbuehl et al, 2010;Pullen et al, 2004), and this result would be consistent with the notion that flagellar membrane proteins traffic to that organelle by a different mechanism than non-membrane proteins. Furthermore, the NPM motif is not obviously represented within the sequence of two other membrane proteins that traffic to the flagellum in Leishmania species, the polytopic aquaporin 1 from L. major (Figarella et al, 2007) and SMP-1, a dually acylated peripheral membrane protein from Leishmania (Tull et al, 2004).…”

Phosphodiesterase Inhibitors as a New Generation of Antiprotozoan Drugs: Exploiting the Benefit of Enzymes that are Highly Conserved Between Host and Parasite

“…PDEB1 and PDEB2 are two tandemly arranged genes that code for two highly similar, cAMP-specific PDEs. Despite their high similarity, their subcellular localization is quite distinct, with TbrPDEB1 localized predominantly in the flagellum, whereas TbrPDEB2 is mainly located within the cell body [64-68]. PDEC codes for a dual-substrate PDE that contains a FYVE domain at its N -terminus [69,70].…”

“…From these, only TcrPDEC has been validated as a therapeutic target [76]. The enzyme has been described by Kunz et al as an unusual dual-substrate PDE [64]. It is able to hydrolyze cAMP as well as cGMP, although the biological significance of the latter activity is unclear.…”

Cyclic-nucleotide signalling in protozoa

“…Two alleles for TbrPDEB2 were identified in the strain used in the protein localization studies, but allelic variation was found not to be responsible for the dual localization (Kunz et al ., 2009). Instead, the N‐terminal 70 amino acids of the two proteins appear to contain the signal for localization, with that from PDEB1 being sufficient to result in specific transport of a green fluorescent protein (GFP) fusion protein to the PFR, whereas the same section from PDEB2 yielded localization mainly in the cell body (Luginbuehl et al ., 2010). Mutating a specific leucine residue to cysteine, and vice versa, resulted in a switching of the localization of the GFP fusion proteins.…”