Assessment of water channel heterotetramerization

CINTIA JOZEFKOWICZ; PABLO ROSI; LORENA SIGAUT; FLORENCIA SCOCHERA; LIA PIETRASANTA; GABRIELA AMODEO; KARINA ALLEVA

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Aquaporin heterotetramerization is emerging as an important point of regulation and fine-tuning of water transport. Many evidences in the literature point to the existence of PIP aquaporins (plant plasma membrane water channels) as heteromeric tetramers formed by a combination of PIP1 and PIP2 isoforms [1-4]. The assemble of heteromers is frequently studied by straightforward functional assessment, generally by means of cRNA co-injection of different PIP isoforms in an heterologous system like Xenopus oocytes followed by water transport measurements, i.e. Pf (osmotic water coefficient) determination. Many results show that Pf of oocytes expressing PIP1-PIP2 heterotetramers is different from PIP2 homotetramers. However neither the threshold of Pf representing an authentic PIP interaction nor the structural elements involved in the interaction are well established. The aim of this work is to accurately analyzed Beta vulgaris PIP aquaporins (BvPIP1;1, BvPIP2;1 and BvPIP2;2) interaction by a combination of water transport assays, pH dose-response experiments and deconvolution of fluorescence confocal microscopy images of PIPs tetramers localization. Interestingly we found that not all PIP2 are able to interact with any PIP1, since at least BvPIP2;1 is unable to promote the incorporation of BvPIP1;1 in the plasma membrane. Strikingly the first extra-cytosolic loop, named loop A, seems to be relevant for PIP1-PIP2 interaction since mutations in that loop modify the behavior of BvPIP2;1-BvPIP1;1 co-expression.