Localization of an Ascorbate-Reducible Cytochrome b561 in the Plant Tonoplast  

Abstract

POB 521, Hungary (A.B.)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 ␣-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.Ascorbate (ASC) plays a key role in the control of growth, development, and defense responses (Davey et al., 2000; Arrigoni and De Tullio, 2002; Mittler, 2002; Pastori et al., 2003). Its role is implied in such diverse processes as the control of cell division and expansion, regulation of programmed cell death, and the regulation of the biosynthesis of ethylene and gibberellic acid. ASC biosynthetic pathways are well characterized in animals and are gradually being elucidated in plants and fungi (Smirnoff, 2001; Smirnoff et al., 2001). In contrast, the cellular mechanisms of ASC regeneration and breakdown remain poorly understood, in particular at the organelle level. The importance of ASC recycling is illustrated by the overexpression of a dehydroascorbate reductase in maize (Zea mays) and tobacco (Nicotiana tabacum), resulting in 2-to 4-fold increased levels of ASC (Chen et al., 2003).A class of membrane proteins, cytochromes b561 (Cyts b561), in plant and animal cells catalyzes transmembrane electron transfer with ASC as the electron donor, thereby contributing to ASC-mediated redox metabolism (Njus and Kelley, 1993; Asard et al., 2001). Members of this protein family use monodehydroascorbate as an electron acceptor, thereby regenerating fully reduced ASC. For example, the chromaffin granule Cyt b561 in the adrenal gland mediates intravesicular ASC regeneration, supporting the biosynt...

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…The reaction was thought to be catalyzed by a cytochrome b561 of the PM (Asard et al, 2001) in analogy with the system for ascorbate regeneration in chromaffin vesicles of mammals (Kelley and Njus, 1986). This concept needs revision, however, because true cytochrome b561 (CYBASC1) is localized in vacuolar membranes (tonoplast) in both Arabidopsis (Griesen et al, 2004) and bean (Preger et al, 2005), and chromaffin membranes are certainly closer to plant vacuolar membranes than PM. Only CYBASC1 of wild watermelon was found targeted to the PM in GFP construct experiments with onion epidermal cells (Nanasato et al, 2005).…”

“…However, the localization in vivo of plant cytochrome b561 is controversial. Arabidopsis CYBASC1 was found to be associated with the tonoplast membrane (Griesen et al, 2004) and annotated in proteomic studies as either a tonoplast protein (Carter et al, 2004;Shimaoka et al, 2004) or a chloroplast protein (Zybailov et al, 2008). Tonoplast localization was also reported for bean (Phaseolus vulgaris) CYBASC1 in etiolated hypocotyls (Preger et al, 2005), whereas a GFP construct of CYBASC1 from wild watermelon (Citrullus lanatus) was shown to be targeted to the PM in transformed onion (Allium cepa) epidermal cells (Nanasato et al, 2005).…”

“…In addition, the apoplastic MDA radical can be reduced back to ascorbate by a trans-PM redox system that uses cytosolic ascorbate as a reductant and involves a highpotential cytochrome b (Horemans et al, 1994). The latter has escaped molecular identification thus far Bérczi et al, 2003;Griesen et al, 2004;Preger et al, 2005).…”

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

“…It has been observed that recombinant and native plant and mammalian Cyts b 561 have characteristic Asc-reducible spectra (30,33,(41)(42)(43)(44). To confirm this biochemical characteristic for DCytb, the absorption spectra were measured using stripped membrane fractions from yeast cells expressing mouse DCytb (Fig.…”

“…Protein Gels and Western Blot-Proteins were resolved by SDS-PAGE using 12% acrylamide gels. Samples were not heated or boiled before loading on the gels, because this caused Cyt b 561 aggregation and prevented them from penetrating the gel (30,33). After SDS-PAGE, proteins were transferred onto a polyvinylidene difluoride membrane with a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad).…”