WATER CHANNELS IN THE PLANT PLASMA-MEMBRANE CLONED BY IMMUNOSELECTIONFROM A MAMMALIAN EXPRESSION SYSTEM

Expression in mammalian COS cells and an efficient microtiter-based st rategy for immunoselection was used in a novel approach to identify ge nes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial m ammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thal iana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two sub families related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since th e cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their cosegregation wi th marker enzymes during aequeous two-phase partitioning. Surprisingly , expression in Xenopus laevis oocytes revealed that all five PIP mRNA s coded for Hg2+-sensitive water transport facilitating activities. Th ere had been no previous evidence of the existence of water channels i n the plasma membrane of plant cells and the high diffusional water pe rmeability of the lipid bilayer was considered to be sufficient for wa ter exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly express ed from the small A. thaliana genome.