Journal Article

Water Channel Activity of Radish Plasma Membrane Aquaporins Heterologously Expressed in Yeast and Their Modification by Site-Directed Mutagenesis

Shinobu Suga and Masayoshi Maeshima

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 7, pages 823-830
Published in print July 2004 | ISSN: 0032-0781
Published online July 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch120
Water Channel Activity of Radish Plasma Membrane Aquaporins Heterologously Expressed in Yeast and Their Modification by Site-Directed Mutagenesis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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.

Keywords: Keywords: Aquaporin — Raphanus sativus — Saccharomyces cerevisiae — Site directed mutagenesis — Stopped flow spectrophotometry — Water channel activity.; Abbreviations: DTT, dithiothreitol; EGTA, ethyleneglycol bis(2-amino-ethyl)tetraacetic acid; NIP, NOD26-like intrinsic protein; PIP, plasma membrane intrinsic protein; RsPIP, radish plasma membrane intrinsic protein; RsTIP, radish tonoplast intrinsic protein; TIP, tonoplast intrinsic protein; SIP, small and basic intrinsic protein.

Journal Article.  5368 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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