Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin.
Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N(2)-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH(3) across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS(1)beta1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS(1)beta1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K(d) = 266 nm). GS(1)beta1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH(3), is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.
Masalkar
Pintu
P
Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA.
Wallace
Ian S
IS
Hwang
Jin Ha
JH
Roberts
Daniel M
DM
D051397:Aquaglyceroporins / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism*; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D018517:Plant Roots / Q000201:enzymology*; D025941:Protein Interaction Mapping; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013025:Glycine max / Q000201:enzymology* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D051397:Aquaglyceroporins; D008565:Membrane Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; C045625:nodulin; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen