Functional Analysis of Nodulin 26, an Aquaporin in Soybean Root Nodule Symbiosomes

Rickey L Rivers,Robert M Dean,Grischa Chandy,James E Hall,Daniel M Roberts,Mark L Zeidel

doi:10.1074/jbc.272.26.16256

Abstract

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (Pf). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (Pf) of SM was high, with a value of 0.05 +/- 0.003 cm/s observed at 20 degrees C (mean +/- S.E.; n = 15). Water flow exhibited a low activation energy, was inhibited by HgCl2 (0.1 mM), and exhibited a unit conductance of 3.2 +/- 1.3 x 10(-15) cm3/s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (Pd) was 0.0024 +/- 0.0002 cm/s, and the resulting Pf to Pd ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.

Highlights

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome
In the present report we show that Nodulin 26 (NOD 26) forms an aquaporin channel upon expression in Xenopus
To address a potential aquaporin function for NOD 26 in rhizobia symbioses, we have examined the water and solute transporting activities of NOD 26 in its native membrane, both to define its potential

Summary

Introduction

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. The bacterium becomes enclosed in a specific organelle, the symbiosome [1], within the infected cells of the nodule This organelle is delimited by a membrane of plant origin, known as the symbiosome membrane (SM).. NOD 26 is a member of an ancient family of membrane channel proteins that include bacterial glycerol transporters, aquaporin (AQP) water channels, and various other intrinsic membrane proteins [6, 7].

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Results

Conclusion