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 1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS 1 1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K d ؍ 266 nM). GS 1 1 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.The formation of legume-rhizobia N 2 -fixing symbioses in root nodules leads to the development of a novel organelle known as the "symbiosome," which houses the endosymbiotic rhizobia bacteroids. In mature nodules, the host infected cells are occupied by thousands of symbiosomes, which constitute the major organelle within this specialized cell type. The symbiosome is delimited by the symbiosome membrane (SM), 2 which controls the transport of all metabolites between the symbiont and the plant host. These transport activities include the efflux of the primary metabolic product of nitrogen fixation (NH 3 /NH 4 ϩ ) and the uptake of dicarboxylates as an energy source to support bacterial N 2 fixation (1, 2). Because of the unique role of the SM, a number of host nodulin ("noduleenhanced") proteins are synthesized and localized to this membrane (3, 4), where they serve transport and regulatory functions in the symbiosis.Early studies revealed that nodulin 26 (nod26), a member of the major intrinsic protein (MIP)/aquaporin superfamily, is among the ensemble of soybean SM nodulins produced during symbiosome biogenesis (4 -7). Since the original identification of nod26 over 20 years ago (4), the role of this channel in the symbiosis has been debated. Functional analyses show that nod26 is a multifunctional "aquaglyceroporin" that transports multiple substrates including water, formamide, and glycerol (6, 8). As a symbiosome-specific aquaglyceroporin, nod26 has been proposed to serve as a low energy transport pathway for water as well as osmolytes within the inf...