ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope.

摘要

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395-401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.