Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue

Abstract

.); and Hawaiian Sugar Planters' Association, Experiment Station, Aiea, Hawaii 96701 -1057 (A.M.) Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Befa vulgaris L.ssp. conditiva) root vacuoles. Positive hybridoma clones and subclones were identified 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 antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELlSAs and they inhibited sucrose 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 sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110-to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(lgM)-specific ELlSAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55-to 65-kD band took up ['TIsucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier.

Characteristics of Sucrose Transport and Sucrose-Induced H+ Transport on the Tonoplast of Red Beet (Beta vulgaris L.) Storage Tissue

“…, were able to inhibit SUC transport (Getz et al, 1993) and corresponding protonexport (Table I) to a comparable extent. This, together with successful reconstitution of a 55-kD polypeptide fraction, identified by use of these carrier-specific antibodies, into artificial proteoliposomes that then exhibited Suc transport activity (Thom et al, 1992;Getz et al, 1993Getz et al, ,1994 indicated close coupling between SUC and proton transport, probably mediated by the same carrier.…”

“…Further evidence that Suc-induced medium acidification represented a translocation of Suc coupled to the efflux of H+ was obtained with monoclonal antibodies previously recognized as carrier specific and used for partia1 purification of the carrier, which was subsequently reinserted into artificial proteoliposomes (Thom et al, 1992;Getz et al, 1993Getz et al, , 1994. The monoclonal antibodies TNP5612 and C5,-,., had virtually no inhibitory effect on Suc-induced proton movement compared to mouse normal serum containing samples when applied at comparable dilutions after heat denaturation (Table I).…”

“…Monoclonal antibodies were provided by Dr. J. Grosclaude (TNP,,-,,. Institute National de la Recherche Agronomique, Jouy-en-Josas, France; Getz et al, 1993) and by the antibody service facility of the University of Hawaii in Honolulu (C50-5.3; Thom et al, 1992). Radiochemicals were supplied by Sigma or by Amersham-Buchler (Braunschweig, Germany).…”

“…Uptake assays with isolated vacuoles were performed according to the methods described by Getz et al (1993). In brief, uptake was initiated by addition of 20 pL of ['4C]Suc (74 kBq mL-') containing Suc solutions of appropriate concentrations to 988 pL of vacuole suspension.…”

“…In brief, about 600 g of washed and peeled red beets were cut into pieces (0.5 X 0.5 X 4 cm). Without plasmolyzing the cells as described previously (Getz et al, 1993), the tissue pieces, washed under running tap water, were cut into 0.35-mm-thick slices. Razor blades and slicing apparatus were rinsed with isolation medium before and during the slicing procedure and the tissue slices and liberated vacuoles were collected in isolation medium (0.75 M sorbitol, 20 mM Hepes/KOH or K-phosphate buffer, pH 7.5, with 2.5 mM DTE added freshly before use; osmotic activity of the isolation medium was 900 2 20 mosmol).…”