Substrate Specificity of the H⁺-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.)

Abstract

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H+-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-a-D-glucopyranoside, phenyl-a-D-thioglucopyranoside, and phenyl-a-D-4-deoxythioglucopyranoside (PDTGP; Ki = 67, 180, and 327 micromolar, respectively). The Km for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyla-D-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-a-D-3-deoxy-3-fluorothioglucopyranoside, phenyla-D-4-deoxy-4-fluorothioglucopyranoside, and phenyl-a-D-thioallopyranoside only weakly but competively inhibited sucrose uptake with K1 values ranging from 600 to 800 micromolar, and phenyl-a-D-thiomannopyranoside, phenyl-,6-D-glucopyranoside, and phenylethyl-,8-D-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-a-D-thioglucopyranoside and PDTGP, were transported.The active transport of sucrose into the phloem tissues of higher plants drives the export of photosynthate carbon and energy to nonphotosynthetic tissues. Numerous studies of sucrose uptake into intact tissues suggest that uptake is carriermediated and catalyzed by a H+-sucrose symport (9). Confirmation of this inechanism of sucrose uptake has been ob- ' Supported by a grant from the Deutsche Forschungsgemeinschaft to T.J.B.2 Present address: Biology Department, Francis Marion College, Florence, SC 29501. tained in PM3 preparations from sugar beet leaves (2, 3, 13), Ricinus cotyledons (19), and spinach leaves (18). In these examples, sucrose uptake into PM vesicles is dependent on a pH gradient, alkaline inside, is electrogenic, and is specific for the sucrose molecule. It also has been demonstrated that sucrose uptake induces a transient alkalization of the exterior medium and that the characteristics of sucrose-induced alkalization and pH-dependent sucrose uptake are identical (4,17). The stoichiometry of sucrose to H+ transport is 1:1 (4, 17). At this time, however, equating the mechanism of sucrose uptake in isolated vesicles with that of phloem loading in vivo is not possible.The chemical features on the sucrose molecule that are involved in the specific interaction of sucrose with the protein carrier are critical to the understanding of the molecular mechanism of sucrose transport. Phloem loading in sugar beets (8) and maize...

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

“…The sources for chemicals were as previously stated (Buckhout, 1989;Slone and Buckhout, 1991;Hecht et al, 1992).…”

“…In the first experiments, PMs from the two-phase system were equilibrated in buffer adjusted to pH 7.5, 6.5, or 5.5 in the presence of 1 mM SUC as described in "Materials and Methods." To test whether the pHi had reached equilibrium, pHi was determined by acetate uptake, assuming an intemal vesicle volume of 4 pL mg-' (Tubbe and Buckhout, 1992).…”

“…Transport is driven by the proton motive force established across the PM by the H+-ATPase in vivo and is electrogenic (Bush, 1990; Slone and Buckhout, 1991). The movement of H+ and Suc is tightly coupled with a stoichiometry of 1:l (Slone and Buckhout, 1991), and the carrier is specific for the Suc molecule, although phenylglucosides are also recognized by the carrier (Hecht et al, 1992). Finally, a gene encoding the H+-Suc symport protein was recently identified in spinach leaf cells (Riesmeier et al, 1992).…”

His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

“…Previous investigations of substrate interaction with the symporter suggested the 3-, 4-, and 6-hydroxyls of the glucosyl moiety of sucrose are important for substrate binding, and the fructosyl half contains a hydrophobic surface that is also critical for substrate recognition (33)(34)(35). Given the size differences between the imidazole ring of His-65 and the basic amino acids, it is difficult to picture how their positive charges could hydrogen bond with one of the important hydroxyl groups of sucrose.…”

Substrate Specificity of the Arabidopsis thaliana Sucrose Transporter AtSUC2

“…In plant cell protoplasts or plasma membrane vesicles, sucrose uptake is inhibited by a variety of glucosides (9,10) providing insight into the structural requirements for binding to sucrose transporters. Results indicated that none of the fructosyl hydroxyls of sucrose interact specifically with the transporter (9).…”