Characterization of solute/proton cotransport in plasma membrane vesicles from Ricinus cotyledons, and a comparison with other tissues

Abstract

Plasma membrane vesicles, purified by aqueous two-phase partitioning, were used to investigate the presence of sugar and amino acid carriers in cotyledons and roots of Ricinus communis L. and in roots of red beet (Beta vulgaris L.). Artificial pH and electrical gradients were generated across the plasma membrane, and [(14)C]acetate and [(14)C]tetraphenylphosphonium were used to demonstrate the presence of an internal alkaline pH gradient and an internal negative membrane potential, respectively. In Ricinus cotyledons, uptake of sucrose was more strongly inhibited than that of glutamine by p-chloromercuribenzenesulphonic acid, phlorizin and phenylglyoxal. The sucrose transport system showed a high degree of substrate specificity with only the presence of maltose and phenyl-α-glucoside significantly affecting sucrose uptake; in contrast, the glutamine transport system was inhibited by a number of other amino acids. ΔpH+gDψ-driven glutamine uptake showed saturation kinetics with a K m of 0.35 mol · m(-3). Sucrose and glutamine Δψ-driven uptake was pH dependent with an optimum in the acidic range (pH 6.25) and a decrease at higher pH values. Vesicles obtained from cotyledons and roots of Ricinus showed different transport properties. In the cotyledons, gDH+gDψ-driven transport for both sucrose and glutamine were observed at similar levels; however, in the root tissue, δpH-Δψ-driven glutamine transport was the dominant uptake process. Uptake rates for glucose and fructose were low in the cotyledons whereas, in the roots, glucose and sucrose transport were slightly higher than that of fructose. In vesicles from red beet tissue there was a different uptake profile, with evidence of proton-coupled cotransport systems for sucrose and glucose, but lower uptake of glutamine and fructose. The results are discussed in relation to the reported different pathways for loading and unloading of solutes in these tissues.

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine‐tagged protein

“…Both transporters are also sensitive to SH group inhibitors such as N-ethylmaleimide (NEM) or p-chloro-mercuriphenylsulphonic acid (PCMPS) which has already been described by various authors (Table 4; Delrot and Bonnemain, 1981 ;Williams et aL, 1992).…”

Functional Characterization and Expression Analysis of the Amino Acid Permease RcAAP3 from Castor Bean1

“…It has previously been found that while NEM inhibits solute transport in intact tissue, it has no effect on uptake into castor bean plasma membrane vesicles. It has been suggested that in castor bean there is little direct effect on the permease but, rather, the response is due to an indirect effect on the driving force (Williams et al, 1992;Weston et al, 1994). The His modifier DEPC has been shown to inhibit amino acid uptake into plasma membrane vesicles (Li and Bush, 1990;Weston et al, 1994).…”

“…Transport of solutes across cell membranes is fundamental to this process of partitioning. Studies in castor bean (Ricinus communis) have suggested that transport of amino acids across the plasma membrane is mediated by specific transporters driven by the proton electrochemical gradient (Williams et al, 1992(Williams et al, , 1996Weston et al, 1994Weston et al, , 1995. Our previous studies in roots of castor bean using isolated membrane vesicles indicated that several different amino acid transporters exist (Weston et al, 1995).…”

Investigation of the Calcium-Transporting ATPases at the Endoplasmic Reticulum and Plasma Membrane of Red Beet (Beta vulgaris)

“…Phase-partitioned plasma membrane was prepared according to a modification of the method described by Williams et al (1992). Red beet microsomal protein (33-35 mg) was suspended in 1.6 cm3 of phase suspension medium containing 250 mo1 m-3 sorbitol, 5 mo1 m-3 potassium phosphate buffer (pH 7.0), and 1 mo1 m-3 DTT.…”