Sucrose Binding Protein-Mediated Sucrose Uptake Across the Plant Plasma Membrane


The processes that regulate carbon allocation to various organs and the developing seed directly impact plant development. In nearly all plants, the most important translocated nutrient is sucrose and, thus, understanding the molecular biology, biochemistry, and physiology of sucrose transport is a central problem in plant biology. Sucrose, a chemically inert carbohydrate, is synthesized in the cytosol of photosynthetically active leaves and is actively loaded into the phloem against a concentration gradient for long distance transport. Because of both the complexity of sucrose movement throughout the plant and its importance to growth and development, one might predict multiple transport mechanisms for this compound. Classic experiments, in fact, demonstrate that three distinct modes of sucrose uptake exist. The first mechanism operates at low (<10 mM) external sucrose concentrations and is characterized by non-linear, saturable uptake kinetics. The second mechanism appears to be simple diffusion and is not specific for sucrose. The third mechanism is a protein mediated event but is nonsaturable (up to 50 - 100 mM sucrose) and displays linear uptake kinetics. Physiological characterization of this linear component demonstrated that it was not simple diffusion and that ~43% of sucrose uptake into leaves was mediated by this linear component in the presence of 25 mM sucrose. After these elegant physiological studies, our mechanistic understanding of this linear uptake mechanism has stalled because the protein responsible for this linear uptake of sucrose was not identified.

The advent of functional complementation in yeast for plant membrane transport processes has facilitated the identification and characterization of several transport proteins in plants. Analysis of the sucrose binding protein (SBP) by heterologous expression in yeast demonstrates that this protein mediates sucrose uptake with features that exactly mimic the physiological details of sucrose uptake ascribed to the linear, nonsaturable mechanism(Overvoorde et al.,1995)). The sucrose binding protein was originally identified by photoaffinity labeling of membranes isolated from the soybean cotyledon, a tissue actively engaged in sucrose transport, with radiolabeled HABS, a sucrose analog which inhibits sucrose transport (Ripp et al., 1988). A cDNA for this protein was obtained and sequenced and an antibody was prepared using purified protein (Grimes et al., 1992) ). We demonstrated that the SBP is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Topological characterization of this plasma membrane protein indicates that it is a type II membrane protein which spans the bilayer once and has the bulk of the protein exposed to the exterior of the cell (Overvoorde and Grimes 1994).

The identification of plant membrane transport proteins within a functional context has set the stage for a tremendous increase in our knowledge of plant membrane transport biochemistry in the near future. The next stage in our research is to address the mechanism of SBP-mediated sucrose uptake. This transport process has several kinetic features that distinguish it from other known transport processes and the SBP shares no sequence similarity to known transport proteins. These observations focus our attention on the fundamental importance of determining the biochemical mechanism mediating this unique uptake mechanism. Research addressing the mechanism of SBP-mediated uptake is underway using a) yeast to analyze PCR-generated mutant SBP proteins, b) functional expression and analysis of kinetic details in frog oocytes, and c) detailed EM-level structural analysis of specialized domains of the plasma membrane where the SBP is concentrated. These structures are regions of invaginated plasma membrane and may harbor clues as to the mechanism of SBP-mediated sucrose uptake because of some striking similarities to the potocytotic mechanism that has been described which utilize folate binding proteins concentrated in the caveolae of epithelial cells. The functional biology of this transport mechanism is being addressed using molecular genetic approaches in plants (Arabidopsis and potato). We are quite excited about this research because of its potential to both answer the enigmatic question of what is the functional role of the linear, nonsaturable uptake mechanism in plants as well as to offer basic insight into a novel biochemical mechanism for sucrose uptake across the plasma membrane.