Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation

Abstract

Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

Figure 1

ZmPIP2;1 and ZmPIP2;6 physically interact in BMS suspension cells. A transgenic cell line expressing 6His-cmyc-ZmPIP2;6 under the control of the ubiquitin promoter in the pAHC25 vector was obtained by biolistic bombardment. Proteins in the microsomal fraction of wild-type BMS cells or BMS cells expressing 6His-cmyc-ZmPIP2;6 were solubilized using 0.035% (w/v) octyl-β-D-thioglucopyranoside for 2 h at room temperature on a rotating wheel.,, After centrifugation at 100,000 g for 30 min, the supernatant was subjected to Ni-column purification (Qiagen, Germany) and the proteins separated by SDS-PAGE and analyzed by western blotting using antibodies raised against ZmPIP2;1 or ZmPIP2;6 (raised in our laboratory) or against cmyc (Santa Cruz Biotechnology, CA) as described previously., Lanes 1 and 3: microsomal fraction of wildtype BMS cells (1) or BMS cells expressing 6His-cmyc-ZmPIP2;6 (3); lanes 2 and 4: nickel column-purified proteins from wild-type BMS cells (2) or BMS cells expressing 6His-cmyc-ZmPIP2;6 (4). The ricin B chain homolog was purified due to the presence of a high number of histidine residues at its N-terminus. The antibodies used for the detection are indicated on the left and the identity of the immunodetected bands is indicated on the right.