Potato aquaporins StPIP1;1 and StPIP1;3 do not facilitate the
transport of ammonia across yeast membranes
In order to test the permeability of aquaporins to solutes such as urea
or ammonia, the heterologous expression system S. cerevisiae is
commonly used. Here we investigated whether StPIP1;1 and StPIP1;3 can
channel ammonia across biological membranes thereby potentially directly
contributing to N uptake under N limiting growth conditions (Fig. 4). We
expressed the two StPIP s in a S. cerevisiae deletion
mutant strain (Δmep1-3 (-ura/-leu)). This mutant has a deletion
in all three ammonium transporter (mep) genes and is therefore
unable to grow on medium containing lower than 5 mM ammonium (Marini,
Soussi-Boudekou, Vissers, & Andre, 1997). An additional source of N
(e.g. in form of proline) is essential to ensure its growth. As
PIP1 aquaporins were repetitively shown to traffic to the PM as
PIP1/PIP2 heterodimers and cannot reach the PM membrane when expressed
alone (G. P. Bienert et al., 2014; M. D. Bienert, Diehn, Richet,
Chaumont, & Bienert, 2018; Fetter, Van Wilder, Moshelion, & Chaumont,
2004), StPIPs were either expressed individually or in
combination with ZmPIP2;5 in the heterologous expression system.
To investigate protein localization in S. cerevisiae we used
GFP:PIP-fusion proteins of the respective aquaporin expressed in yeast
cells. Yeast transformed with GFP:StPIP1;1 or GFP:StPIP1;3alone showed a GFP signal in the internal structures, whereas
co-expressed with ZmPIP2;5 GFP was clearly fluorescent in the PM
(Supplementary Fig. S6). Neither yeast cells expressing StPIP1;1or StPIP1;3 alone (Supplementary Fig. S7), nor in combination
with ZmPIP2;5 were able to grow on ammonia concentrations below 5
mM (Fig. 5). The expression of the positive control hAQP8(Saparov, Liu, Agre, & Pohl, 2007) complemented the mutant and allowed
growth on the medium with low ammonium concentration. This led to the
conclusion that StPIP1;1 and StPIP1;3 are not able to channel ammonia.