Localization of an Ascorbate-Reducible Cytochrome B561 in the Plant Tonoplast

• Published in: Plant physiology (Bethesda) 2004-02, Vol.134 (2), p.726-734
• Main Authors: Daniel Griesen, Dan Su, Alajos Bérczi, Asard, Han
• Format: Article
• Language: English
• Subjects: Antibodies; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Ascorbic Acid - metabolism; Biological and medical sciences; Cell Biology and Signal Transduction; Cell membranes; Cytochrome b Group - genetics; Cytochrome b Group - immunology; Cytochrome b Group - metabolism; Cytochromes; Electron Transport - physiology; Enzymes; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Membrane Proteins - genetics; Membrane Proteins - immunology; Membrane Proteins - metabolism; Metabolism; Monoclonal antibodies; Oxidation-Reduction; Perceptual localization; Plant physiology and development; Plants; Protein isoforms; Protein Isoforms - genetics; Protein Isoforms - immunology; Protein Isoforms - metabolism; Proteins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sucrose - metabolism; Tonoplast; Vacuoles - metabolism; Yeasts
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.103.032359

As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes. It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions. Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments. Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts. However, little is known about the function or subcellular localization of this unique class of membrane proteins. We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family. Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO). Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients. Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with α-band absorbance maximum near 561 nm. The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane. These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.