A Plasma Membrane Sucrose-binding Protein That Mediates Sucrose Uptake Shares Structural and Sequence Similarity with Seed Storage Proteins but Remains Functionally Distinct

Abstract

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a M r indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilinlike protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.The development of plant seeds involves the accumulation of carbon and nitrogen reserves in proteinaceous forms that can both withstand desiccation and be utilized as an energy source by the developing embryo during germination. In legume species, these predominant seed storage proteins are found enclosed in membrane-bound organelles known as protein bodies (1, 2). The globulin seed storage proteins generally fall into two main classes: legumin-like and vicilin-like proteins (1, 3-6). Under nonreducing conditions, the legumin-like proteins are found as hexameric complexes with sedimentation coefficients of 11 S. The subunits of these complexes are derived from a precursor peptide containing two domains: an N-terminal acidic ␣ chain and a C-terminal basic ␤ chain. Following proteolytic processing, these domains remain associated through interchain disulfide links. In contrast, the vicilin-like proteins are found as 7 S trimers under nonreducing conditions. The vicilinlike monomers are 50 -70-kDa polypeptides that undergo variable levels of post-translational proteolytic processing (1, 3).X-ray crystallography of the vicilin-like proteins phaseolin (7, 8) and canavalin (9, 10) has permitted the formation of a canonical three-dimensional model for vicilin-like molecules (8). Each monomer consists of two very similar structural domains reflecting a tandem duplication observed at the nucleotide and amino acid sequences. These tandem domains are composed of two structural elements: a compact eight-stra...

The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 Å resolution

“…The primary sequence of SBP has 20±37% identity and 44±61% similarity with the vicilin-class proteins (Overvoorde et al, 1997). This indicates that the tertiary structure of SBP is closely related to those of canavalin and phaseolin and, as shown below, the potential of SBP subunits to assemble into trimers has also been assessed by model building.…”

“…The model of phaseolin was obtained from the PDB (entry 2phl). According to the sequence alignment of Overvoorde et al (1997), also shown in Fig. 3, the polypeptide templates of residues 46±222, 246±321 and 332±421 were extracted from the re®ned cubic model of canavalin.…”

“…This 62 kDa protein is expressed in young leaves, mature phloem and developing cotyledons, and probably mediates non-saturable sucrose uptake in these plant cells (Overvoorde et al, 1996). Although its function is distinct from storage proteins (Overvoorde et al, 1997), we constructed a homology model of SBP based on the vicilin structure, which might possibly be useful in terms of molecular organization.…”

Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily

“…It is now considered likely that such a His cluster, together with an adjacent conserved Glu, may be the binding site for an Mn 2ϩ ion recently found to be the metal present in OXO, at least in those isolated from cereals (39,238,239). A similar combination of modelling and experimental approaches could be made using the structural data from the sugar-binding domain of the bacterial AraC transcription factor (270) and the sequence data from SBP (bicupins) from higher plants (40,219) in order to identify ligands specifically involved in the binding of either mono-or disaccharides in these subgroups.…”

Thioredoxin-Linked Proteins Are Reduced during Germination ofMedicago truncatulaSeeds

“…In M. truncatula, the protein was found only in cotyledons, again perhaps in preparation for the developmental shift to photosynthesis. Two other binding proteins were identified: P54 and a vicilin-related seed protein that possibly functions in Suc binding (Overvoorde et al, 1997). In pea, P54 undergoes further maturation into P16, a possible participant in chromatin condensation induced by dehydration (Castillo et al, 2000), whereas in M. truncatula, the protein seems not to be further processed and presumably only acts in Suc binding.…”