Characterisation of T. brucei 2T1 cell lines expressing N-terminal tagged TbAQP2^R234K mutant.

A) Blue native-PAGE immunoblot of total cell lysates expressing either TbAQP2 (AQP2^WT), N-terminal lysine-to-arginine mutant (AQP2^3K>R), all lysine-to-arginine mutant (AQP2^5K>R) and individual arginine-to-lysine mutants (AQP2^R19K, AQP2^R45K, and AQP2^R54K). Coomassie blue staining of the same lysates was used as loading control. B) Blue native-PAGE immunoblot of total cell lysates expressing the constructs in (A). Coomassie blue of the same fractions was used as loading control. C) Cell lysates from different lysine-to-arginine mutants shown in (A) and (B) were resolved under denaturing conditions (SDS-PAGE) in a 4–12% acrylamide gel and detected with anti-HA antibody by western blotting. The signal of β tubulin was used as loading control. Note the lack of signal from the AQP2^R147K mutant. D) Cell lines expressing a tetracycline-regulated copy of wild type TbAQP2 (AQP2^WT), N-terminal lysine-to-arginine mutant (AQP2^3K>R), all lysine-to-arginine mutant (AQP2^5K>R) or individual arginine-to-lysine mutants (AQP2^R234K) (Alexa Fluor 488; yellow) were co-stained with anti-TbBiP (endoplasmic reticulum) coupled to Alexa Fluor 568 (magenta). DAPI (cyan) was used to label the nucleus and the kinetoplast. Scale bars 5 μm. D) Protein turnover was monitored by cycloheximide (CHX) treatment followed by chase and western blotting. Prior to treatment, cells were either untreated or treated with 25 μM MG132 for 1 hour. Cells were harvested at 0 and 2 hours post-CHX treatment and cell lysates analyses by western immunoblotting. Quantification represents mean ± standard deviation (n = 3 independent replicates), and dotted line represents protein abundance at time 0h. Statistical analysis was conducted using untreated cells at 2 hours as reference group. ** p<0.001, ns = not significant, using a t-test. EC₅₀ values for pentamidine (E) and salicylhydroxamic acid (SHAM) (F) with or without 5 mM glycerol following expression of AQP2^R234K. Statistical analysis was conducted using untreated cells as reference group. ** p<0.001, ns = not significant, using a t test. Note that this is an extended version of Fig 6A and 6B, and the full panel included for comparison. G) Left panel; View from the cytoplasmic face The TMD4-TMD5 loops in each monomer are highlighted. K234 is shown as spheres. Right panel; Structural overview of T. brucei AQP2 homology model. K147 and K234 are shown as spheres. The expanded view of the conformational change observed during TMD simulations on TMD1 and TMD3 as a result of the K147R mutation. Wild type TbAQP2 is shown in green. TbAQP2 displaying the K147R and K234R mutations is shown in light orange.

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