Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules

Abstract

The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembraneassociated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM.The model legume Lotus japonicus forms nitrogenfixing root nodules after infection by Mesorhizobium loti. The bacteria enter the plant cell by endocytosis, leading to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbors the bacteroids and is surrounded by a peribacteroid membrane (PBM) formed from the plant plasma membrane (PM) during endocytosis of the bacteria (for review, see Verma and Hong, 1996;Whitehead and Day, 1997). The PBM develops further by receiving material from the endomembrane system (see Robertson et al., 1978;Verma and Hong, 1996) and finally forms the structural and functional interface between the symbionts (Robertson et al., 1978). The PBM plays a central role in the exchange of compounds between the organisms (see Udvardi and Day, 1997). To fulfill this role, the PBM is supplied with the specific components, like transporters and enzymes, necessary for the symbiotic exchange processes (see Verma and Hong, 1996;Whitehead and Day, 1997). Several of these activities have been characterized by biochemical and biophysical studies. For example, the activity of P-type H ϩ -ATPase (PATPase) pumping protons from the cytoplasm to the peribacteroid space has been characterized (see Blumwald et al., 1985, and refs. therein). Dicarboxylates are supplied from the plant to the bacteroids, and a specific transporter present in the PBM has been characterized (see Udvardi an...

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