FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

*Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5-15, B-1348 Louvain-la-Neuve, Belgium; and
^†Laboratory of Biochemistry and
^‡Laboratory of Biophysics, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved June 13, 2007 (received for review February 8, 2007)

Abstract

Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, P _f. We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell P _f. Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1–PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability.

Footnotes

^§To whom correspondence should be addressed. E-mail: chaumont{at}fysa.ucl.ac.be
Author contributions: E.Z., J.W.B., H.B., M.A.H., and F.C. designed research; E.Z. and M.M. performed research; E.Z. and J.W.B. contributed new reagents/analytic tools; E.Z., J.W.B., M.M., H.B., M.A.H., and F.C. analyzed data; and E.Z., M.A.H., and F.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0701180104/DC1.
Abbreviations:

AQP,

aquaporin;

PIP,

plasma membrane intrinsic protein;

FLIM,

fluorescence lifetime imaging microscopy;

mCFP,

monomeric cyan fluorescent protein;

mYFP,

monomeric yellow fluorescent protein;

ER,

endoplasmic reticulum;

BMS,

Black Mexican Sweet.