Poster: Membrane Transport

Abs # 1216: Functional expression of radish aquaporin isoforms in yeast

Presenter:	Suga, Shinobu , suga-s@agr.nagoya-u.ac.jp
Authors	Suga, Shinobu  (A)   Maeshima, Masayoshi  (A)
Affiliations:	(A): Graduate School of Bioagricultural Sciences, Nagoya University

Aquaporins (a large protein family) have been believed to facilitate the osmotic water transport across plasma and vacuolar membranes in plant tissues. We previously cloned eight radish aquaporins consisting of 6 plasma membrane aquaporins (PIP) and 2 vacuolar membrane aquaporins (TIP). In radish, the mRNA levels of the PIP2s and the protein level of RsPIP2-1 varied with the tissue, stage of growth and environmental conditions, while the protein and mRNA levels of PIP1s and TIPs were relatively constant (Planta 2001, 212:294-304, Plant & Cell Physiol. 2002, 43:1229-1237). In this study, we investigated the water channel activity of these aquaporins expressed heterologously in a yeast strain (BJ5459), which lacked vacuolar proteases. BJ5459 had two aquaporin genes AQY1 and AQY2. AQY1 protein was not functional and AQY2 was not expressed normally. The expression of all eight aquaporins in yeast could be detected by immunoblot analysis. The membrane fractions were prepared from yeast cells and were assayed for water channel activity with a stopped-flow spectrophotometer (Applied Photophysics Co., model SX.18MV). PIP2s and TIPs exhibited higher water channel activity than the vector control and a mercurial reagent inhibited the activity. Thus, changes in the level of PIP2s might be reflected in the water transport activity of plasma membranes. This may be related to the physiological control of the water transport activity through regulation of the PIP2s level in radish. In contrast to PIP2s and TIPs, the activity of PIP1s was low or negligible. The results suggest that PIP1 members may be negatively regulated under experimental conditions and that they facilitate transport of other substrates.