Loop A Is Critical for the Functional Interaction of Two Beta vulgaris PIP Aquaporins

Abstract

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.

Heteromerization of PIP aquaporins affects their intrinsic permeability

“…Here, we show that when FaPIP1;1 and FaPIP2;1 are coexpressed, the interaction promotes a change in pH sensing as shown by a modification of the observed EC 50 value. Similar results were previously obtained for BvPIPs (21,32). We demonstrate that coexpressing FaPIP1;1 with FaPIP2;1 or FaPIP2;1N228D produced different maximal P f .…”

“…Because it is well established that the MIP quaternary structure is tetrameric (18, 19, 38, 39), all models assumed that all expressed aquaporins form tetrameric structures. Moreover, recent discoveries indicate that PIP interaction occurs via heterotetramerization (25,32).…”

“…These PIP1 fail to reach the PM when expressed alone, but they can succeed if they are coexpressed with PIP2. It has been proposed that this process is a consequence of a physical interaction between PIP1 and PIP2, as reported in both homologous (31) and heterologous systems (14,32). Although there are some PIP1 with the ability to reach the PM on their own (20,27,29), this PIP1-PIP2 interaction seems to be present for several pairs of PIP among different species with functional consequences (14,21,28,(33)(34)(35).…”

“…Recently, it was shown that the first extracellular loop of PIP2 (loop A in BvPIP2;1) could be relevant to the formation of heterotetramers with PIP1 (32). The modification of cytosolic pH sensing, reflected by a shift in the EC 50 of oocytes coexpressing BvPIP2;2 and BvPIP1;1 compared with BvPIP2;2 expressed alone, favors the heterooligomerization hypothesis (21).…”

Two aquaporins, multiple ways of assembly

“…However, while most PIP1 protein is retained in the endoplasmic reticulum unless co-expressed with PIP2, PIP2 is effectively transported to the plasma membrane. [3][4][5][6] The routing modification of PIP1 from the endoplasmatic reticulum to the plasma membrane after its co-expression with PIP2 is due to PIP1-PIP2 physical interaction 3,5 and although this interaction is well supported by experimental evidences, the biophysical aspects and the molecular mechanisms of this event have been identified only recently. 7,8 We investigated the full characterization of the biological and biophysical properties of the different hetero-oligomeric configurations formed by red beet PIP1 and PIP2 subunits.…”