Masters Theses

Date of Award

12-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Elizabeth E. Howell, Jim Hall

Abstract

Infection of soybean roots by nitrogen fixing Bradyrhizobium japonicum leads to expression of plant nodule-specific genes known as nodulins. Nodulin26 is a major component of the symbiosome membrane that encloses the rhizobium bacteroid. Nodulin 26 constitutes 10-15% of the total symbiosome membrane protein and is a member of the aquaporin water channel family. Expression of nodulin 26 and the phosphorylation site mutants S262A and S262D in Xenopus laevis oocytes resulted in a 4-fold increase in osmotic water permeability (Pf) over control uninjected oocytes. The addition of 3 mM HgCl2, a known water channel blocker, reduced the Pf to the levels of control oocytes and this inhibition was reversed upon addition of 5 mM β-mercaptoethanol. To further define the biophysical and biochemical properties of nodulin 26 water channels we studied its transport properties in the native symbiosome membrane and in liposomes containing purified, reconstituted nodulin 26. Permeabilities were determined by use of stopped-flow spectrofluorimetry with symbiosome membrane vesicles or nodulin 26 proteoliposomes loaded with the fluorescent indicator dye carboxyfluorescein.

Symbiosome membrane vesicles exhibited a high Pf (0.05 ± 0.003 cm/s, n=15) that was inhibited 92 % upon addition of 0.1 mM HgCl2. This water transport was characterized by a low Arrhenius Ea (3.3 ± 0.4 kcal/mole, n=3) indicating a facilitated means of water transport. Diffusive water permeability (Pd) was 0.0024 ± 0.0002 cm/s (n =3) and the resulting Pf / Pj ratio was 18.3. If water transport is single file through nod26, than the Pf / Pj value of 18.3 suggests 18 water molecules line up single file within the lumen of the pore and estimates the channel length at 49.8 Å. Symbiosome membrane vesicles are also highly permeable to small nonelectrolytes such as formamide and glycerol, but not urea. The formamide and glycerol flux was inhibited 85-90% by HgCh suggesting that these solutes are transported through nodulin 26.

To determine if purified nodulin 26 was capable of mediating the water and solute fluxes observed for symbiosome membrane vesicles, nodulin 26 was purified from soybean symbiosome membranes and was reconstituted into liposomes for permeability measurements by stopped-flow spectrofluorimetry. Liposomes containing nodulin 26 exhibited a high Pf (0.012 ± 0.001 cm/s, n=4) which was 4-fold over control of pure lipid liposomes. This water permeability was characterized by a low Ea (3.5 ± 1.3 kcal/mole, n=3) and was reduced 67 % upon addition of 1 mM HgCl2. Reconstituted nodulin 26 exhibited a single channel conductance of 3.0 ± 2.9 X 10-15 cm3/s (n=2) similar to the calculated value for nodulin 26 in symbiosome membrane vesicles of 3.2 ± 1.2 x 10-15 cm3/s (n=3). This suggests that purified reconstituted nod26 can mediate water flux at levels similar to those served in the symbiosome membrane. This unit conductance value for nodulin 26 is approximately 30-fold lower than the corresponding value for the “archetypal” water channel aquaporin 1 from red blood cells suggesting that nodulin 26 is an aquaporin of modest conductance. Reconstituted nodulin 26 was also able to facilitate glycerol transport 40-fold over control liposomes and this glycerol flux was inhibited 94 % upon addition of 1 mM HgCl2 consistent with the glycerol flux properties of symbiosome membrane vesicles. Nodulin 26 appears to be a multifunctional aquaporin with water and solute transporting capabilities similar to aquaporin 3, the only other water channel shown to facilitate both water and solute flux.

Overall the data show that symbiosome membrane vesicles contain a mercuric chloride sensitive water and solute transporter and that purified reconstituted nodulin 26 possesses the properties of this transporter.

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