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First published online January 15, 2004; 10.1104/pp.103.032359 Plant Physiology 134:726-734 (2004) © 2004 American Society of Plant Biologists Localization of an Ascorbate-Reducible Cytochrome b561 in the Plant Tonoplast1,2Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (D.G., D.S., A.B., H.A.); and Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, 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
Cyts b561 are widespread in the animal and plant kingdoms, and multiple isoforms are identified in any given species (Asard et al., 2001
Cyts b561 are generally believed to be composed of five or six membrane-spanning Knowledge about the subcellular localization of proteins provides potentially important information to unraveling their physiological function. To address the localization of one of the Arabidopsis Cyt b561 isoforms (cyt b, ASC-dependent [CYBASC1]), we generated antibodies against a C-terminal synthetic peptide and performed membrane fractionation experiments. Our results demonstrate that CYBASC1 is located in the plant vacuolar membrane. These results shed new light on the possible role of ASC in the regulation of the redox status of this organelle.
Generation of CYBASC1-Specific Antibodies and Expression in Yeast
Four putative members of the ASC-reducible Cyt b561 family of trans-membrane proteins (CYBASC1–4) have been identified in Arabidopsis (Asard et al., 2001 To screen for isoform-specific antibodies in the sera from rabbits injected with the CYBASC1 peptide, yeast cells were transformed with cDNAs encoding each of the Arabidopsis Cyt b561 isoforms. Two different annotations are available for the CYBASC1 gene, with translation start sites that are 123 bp apart (protein accession no. NP_567723 [239 amino acids] and accession no. CAA1869 [280 amino acids]). Both CYBASC1 cDNAs were transformed into yeast. cDNAs were cloned downstream of the GAL10-inducible promoter and in-frame with a C-terminal FLAG epitope. Gal treatment of the yeast cultures induced the expression of proteins crossreacting with the FLAG antibody, with molecular masses comparable with the predicted molecular masses of each isoform plus the spacer residues and FLAG epitope (Fig. 1A). Yeast cells transformed with the cDNA for the long CYBASC1 version expressed the short and long protein versions.
Affinity-purified polyclonal CYBASC1 peptide antibodies crossreacted only with proteins from yeast transformed with the CYBASC1 cDNAs (Fig. 1B). The proteins recognized by the CYBASC1 and FLAG antibodies have identical molecular masses, demonstrating that the peptide antibodies recognize the CYBASC1 gene product. In addition, preincubation of the CYBASC1 antibodies with the synthetic C-terminal peptide completely abolished crossreaction of the CYBASC1 antibodies with the recombinant yeast proteins, but did not affect crossreaction with the FLAG antibodies, supporting the specificity of the antibodies (not shown).
To confirm the Cyt nature of the recombinant CYBASC1, absorbance spectra were recorded using membrane fractions from yeast transformed with the CYBASC1 cDNA not containing the FLAG epitope. Reduced-minus-oxidized difference spectra demonstrate the presence of an ASC-reducible b-type cyt with an asymmetrical
Taken together, these results demonstrate that CYBASC1 can be successfully expressed in yeast, and that this gene codes for an ASC-reducible b-type Cyt. Moreover, polyclonal CYBASC1 peptide antibodies specifically crossreact with this protein.
To address whether the CYBASC1 antibodies also recognize an ASC-reducible b-type Cyt in plant membrane fractions, we tested its crossreactivity with purified Cyts from Arabidopsis. Partially purified ASC-reducible Cyts were prepared by solubilization and Mono-Q anion-exchange chromatography of proteins from PM-enriched fractions as described (Bérczi et al., 2003
Mono-Q separations of PM proteins typically result in the separation of two ASC-reducible b-type Cyt peaks, with the first peak containing the majority of the Cyts (not shown; Bérczi et al., 2003
As previously observed (Bérczi et al., 2003
The crossreaction of the CYBASC1 antibodies with proteins derived from a PM-enriched fraction was suggestive for their subcellular localization. However, the observation that only a minor portion of the total ASC-reducible Cyts b was recognized prompted further investigation. Arabidopsis leaf microsomal membranes were fractionated using Suc density gradient centrifugation. The distribution of organelles was determined using marker enzyme activities, antibodies against organelle-specific proteins, and measurement of chlorophyll concentrations. Crossreactivity of the CYBASC1 antibodies in the Suc gradient fractions showed a maximum between 28% and 30% (w/v) Suc (fractions 19–22; Fig. 4). The only profile that correlated closely with the CYBASC1 distribution was that of the TO marker (V-PPiase), with a maximum at similar Suc densities, suggesting a localization in the vacuolar membrane. The PM marker (H-ATPase antibody) showed a distribution with maximum at 34% to 38% (w/v) Suc, and the distribution of Golgi membranes (JIM84 antibody) showed a broad maximum between 30% and 37% (w/v) Suc. A broad maximum for the Golgi fraction was also observed when latent IDPase activity was measured as the Golgi marker (not shown). Chloroplast membranes are located at 38% to 42% (w/v) Suc. The two markers used for mitochondrial membranes (Cyt c oxidase [CCO] activity and F0F1-ATPase antibody) showed overlapping profiles with a maximum around 36% to 42% (w/v) Suc. Endoplasmic reticulum membranes showed a wide distribution as demonstrated by two independent markers (Cyt c reductase activity and Sec12 antibody).
To independently confirm the presence of an ASC-reducible Cyt b in the TO, absorbance spectra were measured in the gradient fractions. Maximal levels of ASC-reducible b-type Cyts were obtained in fractions 22 to 18 (27%–31% [w/v] Suc; Fig. 4), correlating with the fractions with maximal levels of the TO marker. Cyt reduction levels by ASC reached 60% (fraction 20) to 71% (fraction 19) of the reduction levels obtained with ASC + dithionite. Specific activities of ASC-reducible Cyts are 156 and 131 pmol mg–1 protein in the peak fractions, 18 and 19, respectively. Therefore, spectral data, and results with the CYBASC1 antibodies in the Suc density gradient fractions, support the presence of ASC-reducible Cyts b in the TO.
To provide further evidence for the TO localization of ASC-reducible Cyts b, the crossreaction of the CYBASC1 antibodies was compared in a crude microsomal membrane fraction (MF), a PM-enriched fraction, a PM-depleted intracellular membrane (IM) fraction, and a TO-enriched membrane fraction. Antibodies against organelle-specific proteins were used to evaluate the enrichment and contamination levels in these preparations. The crossreaction of the CYBASC1 antibodies was strongly enriched in the TO-enriched fraction when compared with the MF fraction, and this enrichment correlated with that of the TO marker (Fig. 5). As expected, the crossreaction of the H-ATPase antibodies was strongly enriched in the PM-enriched fractions. The Golgi-specific antibodies (JIM84) crossreacted most prominently with the MF and IM fractions, but some crossreaction was observed in the PM- and TO-enriched fractions. Proteins crossreacting with the ER marker antibody (Sec12) were found in the MF and IM fractions and appeared slightly enriched in the TO fraction.
Absorbance spectra demonstrate the presence of an ASC-reducible Cyt b in the TO-enriched membrane fraction (Fig. 6). The
Cyts b561 constitute a newly identified class of proteins in plants and animals involved in ASC-mediated trans-membrane electron transfer (Beers et al., 1986 ; Kent and Fleming, 1987; Asard et al., 2001; Verelst and Asard, 2003). Mammalian Cyts b561 play a role in ASC regeneration, supporting neuropeptide hormone biosynthesis, and possibly in the ASC-mediated reduction of iron chelates (Wakefield et al., 1986; Njus et al., 1987; McKie et al., 2001). Therefore, Cyts b561 are important mediators of ASC-controlled physiological phenomena.
Based on primary sequence conservation and predicted structural features, four putative Cyt b561 genes have been identified in Arabidopsis (Asard et al., 2001
Little is known about the tissue distribution and subcellular localization of the Cyts b561 in Arabidopsis. Plant PM preparations have been demonstrated to contain a Cyt b561 with ASC-mediated transmembrane electron transport activity (Asard et al., 1992
Leaf membranes were fractionated by Suc density gradient centrifugation to identify the subcellular localization of CYBASC1. The crossreactivity of the CYBASC1 antibodies correlated with the distribution of a TO marker, but not with markers for any of the other organelles, suggesting a TO localization for CYBASC1. The protein recognized by the CYBASC1 antibody was also strongly enriched in a TO-enriched membrane fraction. Further evidence for the TO localization of ASC-reducible Cyts b561 was provided by spectroscopic analysis. ASC-mediated reduction of b-type Cyts correlated with the TO marker profile on Suc gradients. Moreover, TO-enriched membrane fractions contain an ASC-reducible b-type Cyt with an
Four genes with highly conserved Cyt b561 properties have been identified in Arabidopsis (Asard et al., 2001
We observed that the CYBASC1 antibodies crossreact with a minor protein derived from a PM-enriched membrane fraction, which, at first sight, appears contradictory with the TO localization for CYBASC1. However, PM fractions, even when purified by aqueous two-phase partitioning, are, at best, "highly enriched" and still contain small amounts of other membrane types, as well as cytoplasmic proteins (Bagnaresi et al., 2000 Two annotations for the CYBASC1 gene are currently available in the database. The molecular mass of the protein recognized by the CYBASC1 peptide antibodies in Arabidopsis corresponds more closely to the short version of the protein, suggesting that the short gene product represents the mature protein.
It becomes increasingly clear that plant vacuoles are more diverse and specialized than has initially been assumed (Paris et al., 1996
TO Cyts b561 are likely to participate in ASC-mediated trans-membrane electron transport, possibly supporting ASC regeneration, as is the case for the mammalian Cyt b561 located in catecholamine secretory vesicles. Plant vacuoles contain high levels of ASC (Rautenkranz et al., 1994 Trans-membrane electron transport mediated by mammalian Cyts b561 occurs from cytoplasmic ASC to an electron acceptor in an acid compartment, i.e. secretory vesicles for the chromaffin granule Cyt b561, or the intestinal lumen for the duodenal Cyt b561. By homology, the TO Cyt b561 is likely to transport electrons from cytosolic ASC to electron acceptors in the acidic vacuolar lumen, consistent with a function in ASC regeneration or iron reduction.
Plant Material and Membrane Preparation Arabidopsis (ecotype Columbia) was grown in a soil/vermiculite mixture (Metro Mix 360; Scots-Sierra, Marysville, OH) at 23°C, 300 µmol m–2 s–1 photosynthetic photon flux density, 50% relative humidity, and 8-h/16-h light/dark cycles. Leaves from 7-week-old plants were harvested on ice and were used for the preparation of membrane fractions. MF were prepared by mixing the plant tissue in a Waring-type blender with six pulses of 5 s and 1-min intervals in ice-cold homogenization buffer (10 mM HEPES, pH 7.5 with KOH, 2 mM EDTA, 1 mM dithiothreitol, 1% [w/v] polyvinyl polypyrrolidone, and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]). Typically, 35 g of fresh weight was homogenized in 200 mL of buffer. The homogenate was squeezed through four layers of cheesecloth, and was centrifuged at 9,000g for 15 min to remove unbroken cells. The supernatant was spun at 48,000g for 60 min to collect membranes, and was resuspended in homogenization buffer using a glass homogenizer.
PM fractions were prepared by aqueous polymer two-phase partitioning (Bérczi et al., 2003
Microsomal membranes were loaded on a 35-mL linear 20% to 44% (w/w) Suc density gradient in gradient buffer (10 mM Tris, pH 7.5 with HCl, 2 mM EDTA, 1 mM dithiothreitol, and 0.1 mM PMSF). Gradients were centrifuged at 100,000g for 16 h at 4°C (SW 28 rotor; Beckman, Fullerton, CA), and 1-mL fractions were collected.
The presence of endoplasmic reticulum and mitochondrial membranes in the Suc gradient fractions was assayed by measuring antimycin A-insensitive, NADH-dependent Cyt c reductase activity and CCO activity, respectively (Lord et al., 1973
The distribution of IMs was also determined by using antibodies against organelle-specific proteins. To detect PMs, monoclonal antibodies (46E5B11D5) against the PM H+-pumping P-type ATPase (100 kD, 1:1,000) were used (gift from Dr. Wolfgang Michalke, University of Freiburg, Freiburg, Germany; Jahn et al., 1998
The concentration of b-type Cyts in Suc gradient fractions was determined from the reduced-minus-oxidized difference spectra (Asard et al., 1989
The concentration of b-type Cyts in TO-enriched fractions, as well as in yeast MFs, was determined from difference spectra recorded at room temperature in dual-wavelength mode (between 500 and 600 nm and with reference at 601 nm, 1-nm slit width, and 1-nm s–1 scan rate) in 2-mL samples under continuous stirring. Spectra were recorded after oxidation of the sample by ferricyanide (0.5 mM) and subsequently after addition of ASC (20 mM) and dithionite (crystals). To increase the signal-to-noise ratio, 16 scans were averaged. Cyt amounts were calculated from the reduced-minus-oxidized difference spectra using the absorbance of the
Antibodies were generated against the 21-amino acid C-terminal peptide ([Cys]-SPSPSPSVSNDDSVDFSYSAI) of CYBASC1 (accession no. NP_567723 and At4G25570). Two rabbits were injected with the KLH-coupled peptide (Cocalico Biologicals, Reamstown, PA). After screening the sera for the presence of CYBASC1-specific antibodies against yeast recombinant CYBASC1, antibodies were affinity purified. The peptide was coupled to agarose using Sulfolink Coupling Gel (Pierce Biotechnology, Rockford, IL). Five milliliters of antiserum was incubated with 1 mL of peptide-coupled agarose and was eluted with 3 mL of ImmunoPure Gentle Ag/Ab Elution buffer (Pierce) after washing with 10 column volumes of Tris-buffered saline (20 mM Tris and 137 mM NaCl, pH 7.5), including 1 M NaCl. Purified antibodies were dialyzed overnight in 1 L of Tris-buffered saline to remove the elution buffer.
Proteins in the membrane fractions were resolved by SDS-PAGE electrophoresis according to Laemmli (1970
Samples were not heated or boiled before loading on the gels because this caused the proteins recognized by the CYBASC1 antibody to aggregate, preventing them from penetrating the gel. A similar observation has been made with the chromaffin granule Cyt b561 from bovine (Duong and Fleming, 1982
Standard PCR methods were used to amplify the genes encoding the Cyt b561 isoforms (CYBASC1–4) from Arabidopsis mixed tissue total RNA. The cDNA sequences for CYBASC1, CYBASC2, and CYBASC4 are, respectively, represented by NM_118689.1, AF132115, and NM_102375.1. For CYBASC3, the annotation previously presented (Asard et al., 2001
For transformation, yeast cells (strain YPH499, ura3-52 lys2-801amberade2-101ochre trp1- Cells were collected by low-speed centrifugation (5,000g for 10 min) when the OD600 reached 0.8, and they were washed in ice-cold homogenization buffer (50 mM MES-KOH, pH 6.5, 5 mM EDTA, 100 mM NaCl, and 100 mM Suc). Washed cells were resuspended in 18 mL of ice-cold homogenization buffer, supplemented with protease inhibitors (1 mM PMSF and 1 µg mL–1 each aprotinin, pepstatin, leupeptin, antipain, and chymostatin), and 0.1% (w/v) ASC. Cells were broken in a Bead Beater (Biospec Products, Bartlesville, OK) by four 1-min cycles with 2-min intervals using 0.5-mm glass beads. The homogenate was centrifuged at 5,000gmax for 10 min at 4°C to remove unbroken cells and heavy membrane vesicles. The microsomal membrane fraction was obtained after centrifugation at 75,000gmax for 60 min at 4°C. The pellet was resuspended in 25 mM MES-KOH, pH 6.5, containing 1% (w/v) glycerol and was stored at –80°C until use.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
We thank Dr. Eduardo Blumwald for the kind gift of purified Arabidopsis TO membranes, Amy Siekman for general laboratory assistance, and Dr. Julie Stone for helpful discussions. Received August 27, 2003; returned for revision September 23, 2003; accepted October 30, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032359.
1 This work was partially supported by the Hungarian National Science Foundation (grant no. OTKA T–034488).
2 This paper is a contribution of the University of Nebraska Agricultural Research Division (Lincoln); journal series no. 14241. * Corresponding author; e-mail hasard2{at}unl.edu; fax 402–472–7842.
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ABSTRACT
RESULTS
-hydroxylase (Harnadek et al., 1985
63 his3-
