N-terminal lysine residues in the N-terminal cytoplasmic tail are important for protein stability, oligomerisation, and anterograde transport.

A) Left panel; Structural predictions of 3xHAAQP2 generated with i-Tasser, indicating the three N-terminal lysine residues (magenta) mutated in AQP23K>R. The 3xHA tag has been omitted for simplicity. Right panel; Fluorescence microscopy of cells expressing N-terminal HA-tagged wild type AQP2 (AQP2WT) or lysine mutant AQP23K>R. Both proteins are shown in yellow. DAPI (cyan) was used to label the nucleus (N) and the kinetoplast (K). Scale bars, 5 μm. Western blot of cell lysates upon induction with tetracycline are also included. B) EC50 values for pentamidine (left panel) and salicylhydroxamic acid (SHAM) with or without 5 mM glycerol (right panel) following recombinant expression of either AQP2WT or AQP23K>R with a tetracycline-regulated (Tet-on) copy in T. brucei 2T1 bloodstream forms. Multiparametric ANOVA calculated as for Fig 4 (N = 3 independent replicates). C) Cell lines expressing AQP2WT or AQP23K>R (Alexa Fluor 488; yellow) were co-stained with anti-BiP (endoplasmic reticulum marker). All markers were labelled with secondary antibodies coupled to Alexa Fluor 568 (magenta). DAPI (cyan) was used to label the nucleus and the kinetoplast, as indicated in (A). Scale bars, 5 μm. D) Native-PAGE immunoblot of total cell lysates expressing either AQP2WT or AQP23K>R. Coomassie blue staining of the same fractions was used as loading control. The triple aqp-null T. brucei 2T1 cells (ΔAQP) were also included as control. E) Left panel; Protein turnover monitored as in Fig 4 for AQP2WT or AQP23K>R. Cells were either untreated or treated with 25 μM MG132 for 1 h prior to harvest. Cells were harvested at 0 hours and 2 h post-CHX treatment, and lysates analysed by immunoblotting. α–β tubulin was used as loading control. Right panel; Protein quantification representing the mean ± standard deviation (n = 3 independent replicates). Dotted line represents 100% (signal in untreated samples). Statistical analysis was conducted using the signal from untreated cells at 2 h as reference group. ** p<0.001, ns = not significant, using a t test.