STOICHIOMETRY AS A KEY MODULATOR OF PIP PLANT AQUAPORINS BIOLOGICAL ACTIVITY

CINTIA JOZEFKOWICZ; LORENA SIGAUT; AGUSTINA CANESSA FORTUNA ; FLORENCIA SCOCHERA; LUIS GONZÁLEZ FLECHA; LÍA ISABEL PIETRASANTA; NICOLÁS AYUB; GABRIELA SOTO; GABRIELA AMODEO; KARINA ALLEVA

Baltimore, Maryland

Conferencia; 59th Annual meeting Biophysical Society; 2015

Biophysical Society

The specific self-association of membrane channels to form oligomeric assemblies is a biological relevant event, which usually confers functional advantages to biological systems. Evidences strongly support that plant plasma membrane aquaporin (PIP) can physically interact to form oligomers by combining PIP2 and PIP1 monomers; however, the kind of oligomer and/or its stoichiometry has not been experimentally elucidated yet.Along this research, we aim at defining whether aquaporins are able to form functional hetero-tetramers (different PIP monomers organized in a single tetramer) with a given stoichiometry that determines their activity and regulation.To achieve this goal, we examine the functional properties of heterotetramers comprising different PIP2-PIP1 ratios. Our experimental approaches include: i) designing mutants to alter PIP2-PIP1 interaction; ii) performing homo and heterodimeric constructs made of either PIP1 or PIP2, as well as both subunits, 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 heterotetramers with different stoichiometries can be functional in a heterologous cellular system since they are able to assemble by expression of PIP2-PIP1 tandem dimers, and by co-expression of those dimers plus PIP2 or PIP1 monomers. Interestingly, the composition of these heterotetramers can modify water transport activity and pH sensitivity by shifting the EC50 of the inhibitory response. Moreover, the first extracellular loop of PIP2 acts as a crucial structural element to achieve PIP heterotetramerization.Thus, our findings throw light not only on PIP heterotetramerization as a novel regulatory mechanism to adjust water transport across the plasma membrane but also on the stoichiometry of PIP heterooligomers, issues that had been unclear for many years in the biophysical field of plant aquaporins.