Protein sorting and expression of a sucrose-binding protein (GmSBP) and a retrograde vesicle transport protein (GmRVT) in developing soybean seed

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a reevaluation of its biological function. The GmSBP-like proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and beta-conglycinin, established several storage protein-like characteristics for GmSBP. All three proteins had a similar distribution within the endoplasmic reticulum, Golgi, and protein storage vacuole. All three proteins increased in expression during seed development, and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, beta-conglycinin (alpha/alpha' subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter region. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.;Sequence analysis of two proteins from a yeast two-hybrid screen identified three putative transmembrane alpha-helices and a C-terminal dilysine motif. Dilysine motifs are common to proteins that participate in retrograde vesicle transport to the endoplasmic reticulum. Due to their putative role in retrograde transport, we have named these proteins soybean retrograde vesicle transport protein 1 (GmRVT1) and 2 (GmRVT2). Subcellular fractionation suggested that GmRVT1 and GmRVT2 were present within the endoplasmic reticulum, but were also distributed throughout various subcellular compartments that may include Golgi, trans-Golgi, prevacuole, vacuole, and plasma membrane. Immunogold labeling of developing soybean cotyledon placed GmRVT within the ER, Golgi, and protein storage vacuole. The sorting of GmRVT to the protein storage vacuole is atypical for a protein containing a dilysine motif. Localization to the protein storage vacuole may be explained by sequence information other than the dilysine motif that confers transport to the protein storage vacuole directly from the ER or following transport through the Golgi.