Stoichiometry as a key modulator of PIP plant aquaporins biological activity

CINTIA JOZEFKOWCIZ; LORENA SIGAUT; A CANESSA FORTUNA; FLORENCIA SCOCHERA; LUIS GONZALEZ FLECHA; LIA PIETRASANTA; NICOLAS AYUB; GABRIELA SOTO; GABRIELA AMODEO; KARINA ALLEVA

Maryland USA

Congreso; 59th Meeting Biophysical Society; 2015

Biophysical Society

The specific self-association of membrane channels to form oligomericassemblies is a biological relevant event, which usually confers functionaladvantages to biological systems. Evidences strongly support that plant plasma membraneaquaporin (PIP) can physically interact to form oligomers by combining PIP2 andPIP1 monomers; however, the kind of oligomer and/or its stoichiometry has notbeen experimentally elucidated yet.Along this research, we aim at defining whether aquaporins are able toform functional hetero-tetramers (different PIP monomers organized in a singletetramer) with a given stoichiometry that determines their activity andregulation.To achieve this goal, we examine the functional properties ofheterotetramers comprising different PIP2-PIP1 ratios. Our experimentalapproaches include: i) designing mutants to alter PIP2-PIP1 interaction; ii) performinghomo and heterodimeric constructs made of either PIP1 or PIP2, as well as bothsubunits, iii) analyzing PIP location by confocal fluorescence microscopy; iv)measuring water transport in control and inhibited conditions (citosolic acidification);and v) studying PIPs in silico.Results show that PIP heterotetramerswith different stoichiometries can be functional in a heterologous cellularsystem since they are able to assemble by expression of PIP2-PIP1 tandemdimers, and by co-expression of those dimers plus PIP2 or PIP1 monomers.Interestingly, the composition of these heterotetramers can modify watertransport activity and pH sensitivity by shifting the EC50 of the inhibitoryresponse. Moreover, the first extracellular loop of PIP2 acts as a crucialstructural element to achieve PIP heterotetramerization. Thus, our findings throw light notonly on PIP heterotetramerization as a novel regulatory mechanism to adjustwater transport across the plasma membrane but also on the stoichiometry of PIPheterooligomers, issues that had been unclear for many years in the biophysicalfield of plant aquaporins.