IMMUNOLOGICAL EVIDENCE FOR THE EXISTENCE OF A CARRIER PROTEIN FOR SUCROSE TRANSPORT IN TONOPLAST VESICLES FROM RED BEET (BETA-VULGARIS L) ROOT STORAGE TISSUE

Monoclonal antibodies were raised in mice against a highly purified to noplast fraction from isolated red beet (Beta vulgaris L. ssp. conditi va) root vacuoles. Positive hybridoma clones and subclones were identi fied by prescreening using an enzyme-linked immunosorbent assay (ELISA ) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibod y-containing ascites fluids on basal and ATP-stimulated sugar uptake i n vacuoles, isolated from protoplasts, as well as in tonoplast vesicle s, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited s ucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and su garcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 co lumns yielded two major peaks in the 55 to 65-kD regions and in the 11 0- to 130-kD regions of solubilized proteins from red beet root tonopl asts, which reacted positively in immunoglobulin-M(IgM)-specific ELISA s with anti-sugarcane tonoplast monoclonal IgM antibodies. Only recons tituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [C-14]-sucrose with linear rates for 2 min, suggesting th at this fraction contains the tonoplast sucrose carrier.