Water channel activity of radish plasma membrane aquaporins heterologously expressed in yeast and their modification by site-directed mutagenesis.
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 times 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.
Suga
Shinobu
S
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence / Q000502:physiology; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D031224:Raphanus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D019076:Transgenes / Q000235:genetics; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism; D014882:Water-Electrolyte Balance / Q000235:genetics
C115153:AQY1 protein, S cerevisiae; C422454:AQY2 protein, S cerevisiae; D020346:Aquaporins; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins