Chimerisation of TbAQP2 leads to mislocalisation, reduction in glycerol transport activity and rapid turnover.
A) Cell lines expressing N-terminal HA-tagged TbAQP1, TbAQP2, TbAQP3, field-isolate chimeric AQP2/3 (40AT) or a single TMD mutant (AQP2TMD4) (Alexa Fluor 488; yellow) co-stained with anti-ISG75 (magenta). DAPI (cyan) was used to label the nucleus and the kinetoplast. Scale bars 5 μm. Western immunoblotting analysis from lysates of cells expressing these constructs are also included. Anti-β tubulin was used as loading control. B) BN-PAGE immunoblot of total cell lysates expressing the constructs in (A). Coomassie blue staining of the same fractions was used as loading control. C) EC50 values (average ± standard deviation; n = 3) for salicylhydroxamic acid (SHAM) with or without 5 mM glycerol following recombinant expression of the constructs in (A). D) Left panel; Representative western blotting (n = 3 independent replicates) analysis of protein turnover monitored by cycloheximide (CHX) treatment followed by pulse-chase of cells expressing the constructs in (A). Right panel; Protein quantification representing the mean ± standard deviation of three independent experiments. Dotted line represents 50% of protein abundance. Data presented as mean ± standard deviation (n = 3 independent replicates).