BIOTINYLATION OF CELL-SURFACE PROTEINS IN CARROT SUSPENSION CELLS

The ability of sulfosuccinimidobiotin to label proteins associated with the plasma membrane and cell wall of intact carrot suspension cells was addressed. This compound covalently modifies proteins and these biotinylated proteins can be detected after SDS-PAGE. The labeling reaction was dependent upon two critical features: 1) using a phosphate buffer at pH 8.0 and 2) adding the sulfosuccinimidobiotin directly to resuspended cells. Using these optimized conditions, it was possible to detect biotinylated cell surface proteins after SDS-PAGE. The cell surface specificity of this label was confirmed with sucrose gradient techniques, aqueous phase partitioning, and cell wall protein extraction. The data also suggest that enzymatic removal of the cell wall results in the removal and/or alteration of potential labeling sites on the plasma membrane.