Water channel activity of radish plasma membrane aquaporins heterologously expressed in yeast and their modification by site-directed mutagenesis

• Published in: Plant and cell physiology Vol. 45; no. 7; pp. 823 - 830
• Main Authors: Suga, S. (Nagoya Univ. (Japan)), Maeshima, M
• Format: Journal Article
• Language: English
• Published: Japan Oxford University Press 01.07.2004; Oxford Publishing Limited (England)
• Subjects: Amino Acid Sequence - physiology; Aquaporins - genetics; Aquaporins - metabolism; Cell Membrane - genetics; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell Membrane Permeability - genetics; CELL MEMBRANES; dithiothreitol; DTT; EGTA; ethyleneglycol bis(2-amino-ethyl)tetraacetic acid; GENE EXPRESSION; Gene Expression Regulation, Fungal - genetics; Gene Expression Regulation, Plant - genetics; Keywords: Aquaporin — Raphanus sativus — Saccharomyces cerevisiae — Site directed mutagenesis — Stopped flow spectrophotometry — Water channel activity; Molecular Sequence Data; Mutagenesis, Site-Directed - genetics; MUTANTS; MUTATION; NIP; NOD26-like intrinsic protein; PERMEABILITY; Phosphorylation; PIP; PLANT WATER RELATIONS; plasma membrane intrinsic protein; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Structure, Tertiary - physiology; radish plasma membrane intrinsic protein; radish tonoplast intrinsic protein; Raphanus - genetics; Raphanus - metabolism; Raphanus - ultrastructure; RAPHANUS SATIVUS; RsPIP; RsTIP; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sequence Homology, Nucleic Acid; SIP; small and basic intrinsic protein; TIP; tonoplast intrinsic protein; Transgenes - genetics; Vacuoles - genetics; Vacuoles - metabolism; Water-Electrolyte Balance - genetics
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pch120

Plants contain a number of aquaporin isoforms. We developed a method for determining the water channel activity of individual isoforms of aquaporin. Six plasma membrane aquaporins (RsPIPs) and two vacuolar membrane aquaporins (RsTIPs) of radish (Raphanus sativus) were expressed heterologously in Saccharomyces cerevisiae BJ5458, which is deficient in endogenous functional aquaporin. Aquaporins were detected by immunoblot analysis with corresponding antibodies. Water permeability of membranes from yeast transformants was assayed by stopped-flow spectrophotometry. The water channel activity of members of the RsPIP2 and RsTIP subfamilies was about 10 times and 5 tunes greater, respectively, than that of the control; however, RsPIP1s had little (RsPIP1-2 and RsPIP1-3) or no activity (RsPIP1-1). Site-directed mutation of several residues conserved in RsPIP1s or RsPIP2s markedly altered the water transport activity. Exchange of Ile244 of RsPIP1-3 with valine increased the activity to 250% of the wild type RsPIP1-3. On the other hand, exchange of Val235 of RsPIP2-2, which corresponds to RsPIP1-3 Ile244, with isoleucine caused a marked inactivation to 45 % of the original RsPIP2-2. Mutation at possible phosphorylation sites at the N- and C-terminal tails also altered the activity. These results suggest that these residues in the half-helix loop E and the tails are involved in the water transport and the functional regulation of RsPIP1 and RsPIP2.