Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins

Abstract

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO 2 -triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO 2 -related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO 2 -triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO 2 -related transport processes, a structure built up by the tetramer is the basis of this function.Water moves across biological membranes by diffusion. In most living organisms, the rate of water diffusion can be increased via pore-forming transmembrane proteins, the socalled aquaporins. These consist of six-membrane-spanning helices; N and C termini of the proteins face the cytosol. The helix-connecting loops B and E are themselves short helices that dip into the membrane from opposite sides and form the water-conducting channel (1, 2). Aquaporin monomers can assemble into tetramers (3, 4). Evidence for their function as water transport facilitators was observed by results from experiments using heterologous expression systems, such as Xenopus laevis oocytes (5). Despite this initially detected function, a facilitated membrane transport for glycerol or volatile substances like CO 2 or NH 3 was postulated (6 -9). Plant aquaporins were subdivided into protein groups according to the cellular location in which they have been initially detected and on the basis of sequence similarities (10). Accordingly, the PIPs (plasma membrane-intrinsic proteins) were split into two major groups, the PIP1 and PIP2 aquaporins. Compared with PIP1 proteins, PIP2 proteins have a shorter N-terminal extension and a longer C terminus (11)(12)(13)(14). In general, plant PIP2 proteins have been shown to facilitate membrane water transport in heterologous expression systems; however, PIP1 proteins display low or no activity in this respect (8,11,(15)(16)(17)(18). For the human AQP1 as well as for the tobacco PIP1 aquaporin NtAQP1, an increased cellular acidification rate under CO 2 -enriched buffer was obtained in oocytes expressing the respective aquaporin in addition to a carbonic anhydrase (9,19,20). The observations on NtAQP1, together with the fact that plants deficient in NtAQP1 express...

The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange under Well-Watered Conditions But Not under Water Stress

“…According to the observations made by Vandeleur et al (2009), grapevine VvPIP2;2 requires coexpression of VvPIP1;1 to be fully active as a water channel. A similar interaction of PIP1s with PIP2s has previously been reported (Fetter et al, 2004) and can be due to heteromerization of the aquaporin tetramer (Otto et al, 2010). We observed that VvPIP1;1 was not expressed in leaves, confirming the results of Baiges et al (2001), while it was expressed in roots, in particular in some transgenic lines (4, 28, and 33) characterized by low R h root .…”

Expression Analysis of Aquaporins from Desert Truffle Mycorrhizal Symbiosis Reveals a Fine-Tuned Regulation Under Drought

“…Several studies, such as inhibition with mercury (Terashima and Ono 2002) and transgenic plants with altered AQP expression systems (Flexas et al 2006;Hanba et al 2004;Uehlein et al 2003Uehlein et al , 2008, have demon- strated that PIP1 proteins can alter leaf mesophyll conductance to CO 2 (g m ) and assimilation capacity. One of the most studied cooporins is NtAQP1 from tobacco, which also increases CO 2 conductivity when expressed in yeast (Otto et al 2010). Given that HaPIP1;1 enhances the CO 2 permeability of yeast membranes, we would expect it to increase g m in H. almeriense leaves which, in turn, would increase leaf intercellular CO 2 concentration and decrease g s .…”

“…Several reports have shown that PIP1 AQP, expressed in heterologous systems, induce very low or undetectable increases in membrane water permeability (Chaumont et al 2000;Kammerloher et al 1994;Otto et al 2010;Secchi et al 2007Secchi et al , 2009). Thus, proteins from the PIP1 family usually exhibit little or no water channel activity when expressed in oocytes, although they have been reported to interact with PIP2 isoforms and to increase their membrane permeability (Fetter et al 2004;Temmei et al 2005;Zelazny et al 2007).…”

Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation

“…Plants have multiple PIP isoforms (13 in Arabidopsis thaliana), and on the basis of sequence similarity, PIPs can be divided into two groups, PIP1 and PIP2. PIPs are thought to occur as tetramers, with each subunit forming an individual aqueous pore, but recent work from several laboratories has indicated that PIP1s and PIP2s can assemble to form heterotetramers (Zelazny et al, 2007;Otto et al, 2010). The function of PIPs in plants has attracted extensive research.…”