Table 1. Summary of the impact of cytoplasmic lysine mutagenesis on TbAQP2 localisation and function.

Protein	Localisation	Protein abundance post-CHX2		Proposed degradation pathway	EC50 pentamidine (nM)3	EC50 SHAM (μM)3	EC50 SHAM + 5 mM glycerol (μM)3
		Untreated	+ MG132
AQP2WT	Flagellar pocket	61.05 ± 3.43%	44.42 ± 13.15%	Lysosome	3.29	1.96	1.16
1AQP2R19K	Endoplasmic reticulum	14.42 ± 9.5%	16.11 ± 7.1%	ERAD1	51.18	1.92	1.12
AQP2R45K	Endoplasmic reticulum	1.3 ± 0.93%	34.3 ± 4.65%	ERAD	43.16	1.99	2.36
AQP2R54K	Endoplasmic reticulum	10.84 ± 0.5%	41.05 ± 12.5%	ERAD	43.10	2.10	2.38
AQP2R234K	Endoplasmic reticulum	7.67 ± 1.9%	24.91 ± 5.12%	ERAD	39.95	1.28	2.34
AQP23K>R	Endoplasmic reticulum	5.16 ± 0.62%	46.18 ± 5.95%	ERAD	27.20	10.10	10.14
AQP25K>R	Endoplasmic reticulum	16.8 ± 6.2%	37.5 ± 9.5%	ERAD	41.98	1.34	1.25

¹AQP2^R19K did not show a significant accumulation upon MG132 treatment, but co-localized with TbBiP, indicating probable ERAD-mediated turnover.

²Protein abundance was calculated 2h post-treatment with cycloheximide (CHX) and expressed as percent of protein abundance compared to protein signal before treatment (“time 0h”).

³Estimated EC₅₀ values from cells induced with tetracycline, 1 μg/ml for 24h.