Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site.

Abstract

The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-ProAla motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, 6-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of 6-TIP is sensitive to mercury. However, the mercurysensitive cysteine residue found in mammalian aquaporins is not present in 6-TIP or in 1-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for 6-TlP and y-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of 651P with y5IP and a-TIP showed that the TlPs are differentially expressed.

Two TIP‐like genes encoding aquaporins are expressed in sunflower guard cells

“…Protein-modifying sulfhydryl reagents such as mercuric chloride are characteristic inhibitors of many aquaporins (Daniels et al, 1996;Maurel et al, 1993;Preston et al, 1992;Yamada et al, 1995). SunTIP7 and SunTIP20 water channel activities in Xenopus oocytes were about 90% …”

“…It showed 70% homology to the Arabidopsis 5TIP sequence (Daniels et aL, 1996) which belongs to the TIP subfamily of MIP. This cDNA clone was used to screen 60000 plaques of a sunflower leaf cDNA library.…”

“…inhibited by 1 mM HgCI 2. In a recent paper, Daniels et al (1996), demonstrated that cysteine residues in transmembrane domain at positions 116 and 118 play a role in the mercury sensitivity of ~TIP and 7TIP, respectively. SunTIP7 and SunTIP20 also contain a cysteine residue at position 116 that is thus also likely to be a mercury-sensitive site (Figure lb).…”

“…The effective location in the membranes has only been demonstrated for a few MIP proteins. A water transport activity has been characterized for some members of both the PIP and TIP subfamilies (Daniels et aL, 1996;Kammerloher et aL, 1994;Maurel et aL, 1993;Yamada et aL, 1995). The expression of plant MIP genes can be regulated at both transcriptional (H6fte et al, 1992) and post-translational (Johansson et al, 1996;Maurel et aL, 1995) levels.…”

“…The expression of plant MIP genes can be regulated at both transcriptional (H6fte et al, 1992) and post-translational (Johansson et al, 1996;Maurel et aL, 1995) levels. This expression can be organ and tissue-specific with a pattern suggesting that aquaporins are involved in both cell expansion and vascular water permeability (H6fte et al, 1992;Jones and Mullet, 1995;Daniels et al, 1996). Water channels are assumed to largely contribute to hydraulic conductivity in Chara cells (Henzler and Steudle, 1995), Arabidopsis leaf protoplasts (Kaldenhoff et al, 1995), the tomato root system (Maggio and Joly, 1995), and sunflower hypocotyls (Hejnowicz and Sievers, 1996).…”