Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV

Abstract

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.

Figure 1

Western blot of GLDH in potato leaf subcellular fractions (A) and mitochondrial inner- and outer-membrane-enriched fractions (B). Fifty micrograms of protein was loaded onto 12% (w/v) denaturing acrylamide gels, transferred to nitrocellulose membranes, and blotted against a specific antibody against GLDH from potato. Px, Peroxisomes; Mit, mitochondria; Mic, microsomes; Cyt, cytosol; Chl, chloroplasts; OM, mitochondrial outer-membrane-enriched fraction; IM, mitochondrial inner-membrane-enriched fraction.

Figure 2

Predicted inner mitochondrial membrane-spanning region of GLDH. GLDH transmembrane regions and orientation were predicted using the Tmpred program. A, GLDH protein sequence from sweet potato (Imai et al., 1998). Areas included in boxes are membrane-spanning regions, while those underlined are peptides generated after digestion with a V8 protease. The Leu at position 137 indicates the normal position for the FAD-binding domain, and is located on the outside of the inner mitochondrial membrane. B, Predicted inner mitochondrial membrane-spanning region of GLDH.

Figure 3

Hypothetical model of the interaction between GLDH and the mitochondrial electron transport chain. GLDH feeds electrons into Cyt c between complexes III and IV, while GL is converted into ascorbate. The inhibition of Cyt c oxidase by KCN in complex IV blocks ascorbate production, possibly by the accumulation of electrons in Cyt c. AOX, Alternative oxidase; DH, dehydrogenase; UQ, ubiquinone.