TOR-induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease

Abstract

TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain which was recognized by TOR in this internalization pathway was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking.

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major

“…To investigate whether the upregulation of uptake mediated by NT1 corresponded to an increase in the level of NT1 protein, immunoblots (Fig. 1B) were performed using an anti‐NT1 antibody directed against the large intracellular hydrophilic loop between transmembrane domains 6 and 7 of the L. mexicana NT1 transporter (Detke, 2007). The level of NT1 protein in isolated membranes of wild‐type parasites was barely detectable.…”

“…Briefly, membrane was incubated with 8–10 ml LI‐COR blocking buffer for 1 h at room temperature with gentle agitation. Subsequently, the membrane was incubated simultaneously with rabbit polyclonal anti Leishmania mexicana NT1‐loop VII (Gln236‐Lys331) antibody (Detke, 2007) and mouse anti α‐tubulin (Calbiochem‐Novabiochem, San Diego, CA, USA) antibodies (dilutions 1:3000 and 1:5000 respectively) in 6–8 ml of LI‐COR blocking buffer overnight at 4°C. The next day, the antibody solution was removed and the membrane was washed four times for 10 min each with 15 ml PBS with 0.1% Tween (PBS‐T) before addition of secondary antibody conjugated to fluorescent dyes, IRDye 800CW Goat Anti Rabbit IgG and IRDye 680 Goat Anti Mouse IgG (dilution 1:20 000, Odyssey, LI‐COR), in 6–8 ml LI‐COR blocking buffer with gentle agitation for 45 min at room temperature.…”

Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner

“…Overexpression of TOR in Leishmania or yeast leads to the redirection of the adenosine transporter from the plasma membrane to the MVT-lysosome and increased resistance to toxic purine analogues internalized by this transporter (Detke, 2007). When the Leishmania adenosine transporter was expressed in yeast, TOR-mediated lysosomal degradation was dependent on the presence of an intact ubiquitination system (Detke, 2007). While indirect, these observations suggest that TOR, which was partially localized to the Golgi apparatus in Leishmania , may have a role in regulating the ubiquitination of the adenosine transporter.…”

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

“…TOR appears to act at the protein level and affect the activities and/or concentration of a class of transporters and other proteins [24]. TUB is transported by adenosine permease, but this purine analog, which is toxic to the parasite, must first enter the cell.…”