Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin — JBC

Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin*♦
Pintu Masalkar‡, Ian S. Wallace‡, Jin Ha Hwang‡§ and Daniel M. Roberts‡§,1
+ Author Affiliations

From the ‡Department of Biochemistry and Cellular and Molecular Biology and
the §Program in Genome Science and Technology, The University of Tennessee, Knoxville, Tennessee 37996
1 To whom correspondence should be addressed. Tel.: 865-974-4070; Fax: 865-974-6306;
E-mail: drobert2@utk.edu.

Abstract
Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N2-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH3 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 GS1β1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS1β1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (Kd = 266 nm). GS1β1 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 NH3, 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.

abstract:
Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin — JBC