Localization of an Ascorbate-Reducible ... - Plant Physiology

Localization of an Ascorbate-Reducible Cytochrome b561 in the Plant Tonoplast1,2 Daniel Griesen, Dan Su, Alajos Be´rczi, and Han Asard* Department 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; Asard et al., 2001). Members of this protein family use monode1 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 [email protected]; fax 402– 472– 7842. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032359.

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hydroascorbate 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 biosynthesis of noradrenaline by dopamine ␤-hydroxylase (Harnadek et al., 1985; Menniti et al., 1986). Similarly, a Cyt b561 in plant plasma membrane (PM) preparations catalyzes trans-membrane electron transport with monodehydroascorbate as an electron acceptor (Asard et al., 1992; Horemans et al., 1994). Although a role in ASC regeneration is apparent for the Cyts b561, it has recently been suggested that these proteins may play a role in iron metabolism. A Cyt b561 from duodenal brush border membranes and the chromaffin granule Cyt b561 were reported to catalyze the reduction of ferric-iron chelates when expressed in Xenopus oocytes (Vargas et al., 2003). Whether plant Cyts b561 also have ferric reductase activity remains to be determined. Cyts b561 are widespread in the animal and plant kingdoms, and multiple isoforms are identified in any given species (Asard et al., 2001, 2002; Verelst and Asard, 2003). Genome analyses have revealed at least three Cyt b561 isoforms in humans and mice (Verelst and Asard, 2003). Two of these are located in distinct cell types and in different subcellular membranes, possibly reflecting their specific physiological roles. Four putative Cyt b561 isoforms have been identified in Arabidopsis. However, little is known about the subcellular localization and physiological function of these proteins. Biochemical evidence has

Plant Physiology, February 2004, Vol. 134, pp. 726–734, www.plantphysiol.org © 2004 American Society of Plant Biologists

Ascorbate-Reducible Cytochrome b561 in the Tonoplast

demonstrated the presence of at least one ASCreducible Cyt b561 in PM-enriched fractions from plants, including Arabidopsis (Asard et al., 1989; Be´ rczi et al., 2001). However, it is not clear which of the four isoforms is represented by this activity. Cyts b561 are generally believed to be composed of five or six membrane-spanning ␣-helices, with two pairs of strictly conserved His residues possibly coordinating two heme molecules (Perin et al., 1988; Degli Esposti, 1989; Srivastava, 1996; Tsubaki et al., 2000; Bashtovyy et al., 2003). In a current model on the structure of these proteins, the two putative heme coordinating His pairs are located on four transmembrane helices (II and IV, and III and V, respectively). This feature distinguishes the Cyts b561 from other trans-membrane b-type Cyts in which the heme-coordinating His residues are located on two helices only (Degli Esposti, 1989). ASC functions as an electron donor to the Cyts b561, and the Michaelis-Menten type kinetics of the ASC-mediated reduction indicate the presence of a specific ASCbinding site (Flatmark and Terland, 1971; Kipp et al., 2001). However, very little is known about the molecular mechanism of electron transport and physiological role of these proteins. 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.

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 GAL10inducible 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 ␣-band with absorbance max-

RESULTS 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; Arabidopsis Membrane Protein Library at http://www.cbs.umn.edu/Arabidopsis/). However, little is known about their subcellular localization and physiological function. Biochemical evidence has demonstrated the presence of an ASC-reducible Cyt b561 in Arabidopsis leaf PM fractions (Be´ rczi et al., 2001, 2003), but it remains unclear which of the isoforms is represented by this activity. To identify the subcellular localization of one of the Arabidopsis Cyts b561, we generated polyclonal antibodies against a C-terminal CYBASC1 peptide. To screen for isoform-specific antibodies in the sera from rabbits injected with the CYBASC1 peptide, yeast cells were transformed with cDNAs encoding Plant Physiol. Vol. 134, 2004

Figure 1. Expression of Arabidopsis Cyts b561 in yeast and crossreaction of the CYBASC1-peptide antibodies with the recombinant protein. A, Western blot of proteins obtained from yeast transformed with Arabidopsis Cyt b561 isoforms (CYBASC1–4) containing the FLAG epitope, probed with a FLAG antibody. The molecular masses of the recombinant proteins closely correspond to the predicted molecular masses for each of the isoforms. B, Western blot of yeast proteins as in A, probed with affinity-purified polyclonal antibodies against the CYBASC1 C-terminal peptide, demonstrating the specificity of the antibodies. Lane 1, Cells transformed with the “empty” vector; lane 2, CYBASC1Long; lane 3, CYBASC1Short (see text for details); lane 4, CYBASC2; lane 5, CYBASC3; lane 6, CYBASC4. Approximately equal amounts of protein (10 ␮g) were loaded in each lane. 727

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Figure 2. Membrane fractions of yeast transformed with the CYBASC1 gene contain an ASC-reducible b-type Cyt. Reducedminus-oxidized absorbance spectra demonstrate the presence of an ASC-reducible b-type Cyt in transformed yeast cells, with a typically asymmetric ␣-band and wavelength maximum near 561 nm. Spectra were recorded after oxidation by ferricyanide and subsequent reduction with ASC or ASC ⫹ dithionite, as described in “Materials and Methods.” Sample protein concentration ⫽ 2.05 mg mL⫺1.

imum near 561 nm (Fig. 2). Reduction by ASC (20 mm) reached about 75% of the ASC ⫹ dithionite reduction. The specific concentration of the ASCreducible Cyt in the 5- to 75,000-g membrane fraction was 1,012 ⫾ 254 (n ⫽ 4) pmol mg⫺1 protein. Importantly, no ASC-reducible b-type Cyts were detected in membrane fractions from yeast transformed with the vector without insert. When CYBASC1 was expressed with the FLAG epitope, ASC-reducible Cyts could often not be detected, or only at much lower levels. This suggests that the epitope interfered with proper protein folding and/or heme incorporation (not shown). 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.

and because a purification protocol for ASCreducible Cyts from PMs is available. 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; Be´ rczi et al., 2003). The CYBASC1 antibodies crossreact with a protein in the second peak (Fig. 3), but not in the first peak (not shown). The protein recognized by the CYBASC1 antibodies has a molecular mass of 28 to 29 kD, closely corresponding to the predicted molecular mass of the short CYBASC1 version (27.5 kD). As previously observed (Be´ rczi et al., 2003), the profiles of the ASC-reducible and dithionitereducible Cyts eluting in the second Mono-Q peak were not completely overlapping. Fraction 27 (Fig. 3) contained the highest total level of Cyts (105 pmol), of which only 15% was ASC reducible, whereas fraction 26 contained 72 pmol of Cyts, of which 47% was reducible by ASC. These data demonstrate the presence of two distinct Cyt b species in this peak, eluting at similar salt concentrations, and one of which is not reducible by ASC (see also Be´ rczi et al., 2003). Importantly, the crossreactivity of the CYBASC1 peptide antibody with the fractionated proteins correlated closely with the presence of ASC-reducible Cyts but not with the dithionite-reducible Cyts. Maximum crossreactivity was observed in the fraction with highest ASC reduction levels (fraction 26), and levels of crossreaction were similar in fractions 27 and 28, as was also observed for the level of ASC reduction (Fig. 3). Our results demonstrate that the CYBASC1 antibodies crossreact with an ASC-reducible Cyt b from Arabidopsis membranes.

CYBASC1 Antibodies Recognize an ASC-Reducible Cyt b in Arabidopsis Membrane Fractions

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 (Be´ rczi et al., 2003). Even though the subcellular localization of CYBASC1 was not known at this point, PMs were an obvious choice because they had previously been demonstrated to contain an ASCreducible b-type Cyt with similarity to Cyts b561, 728

Figure 3. CYBASC1 peptide antibodies crossreact with a partially purified ASC-reducible b-type Cyt from Arabidopsis. Proteins from PM-enriched preparations were solubilized and separated by Mono-Q ion-exchange chromatography. The western blot shows crossreactivity of the CYBASC1 peptide antibodies with the proteins in the corresponding Mono-Q fractions (5 ␮L for each fraction). The level of and crossreactivity of the antibodies correlates closely with the levels of ASC-reducible Cyts. Plant Physiol. Vol. 134, 2004


Localization of CYBASC1 and ASC-Reducible Cyts in Suc Gradients

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 (HATPase antibody) showed a distribution with maximum at 34% to 38% (w/v) Suc, and the distri-

bution 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 ASCreducible 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.

Association of CYBASC1 and ASC-Reducible b-Type Cyts with TO-Enriched Membrane Fractions

Figure 4. The distribution of CYBASC1 correlates with the distribution of the TO in Suc density gradients. Arabidopsis leaf microsomal membranes were fractionated by Suc gradients and intracellular membranes (IMs) were detected by antibodies against organellespecific proteins, marker enzyme activities, and chlorophyll content. Crossreactivity of the CYBASC1 peptide antibodies and the presence of ASC-reducible b-type cytochromes correlate closely with the distribution of the TO marker. Plant Physiol. Vol. 134, 2004

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 ␣-band wavelength maximum of the reduced protein is around 561 nm and shows a very similar asymmetry as observed with the 729

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Figure 5. CYBASC1 is enriched in TO-enriched membrane fractions from Arabidopsis. western blots of microsomal membranes, plasma membranes (PM), IM, and a TO-enriched fraction from Arabidopsis leaves were probed with CYBASC1 peptide antibodies and antibodies against organelle-specific proteins. Crossreactivity of CYBASC1 is strongly enriched in the TO fraction. Protein amounts in all lanes are between 4 and 6 ␮g.

CYBASC1 protein expressed in yeast (see Fig. 2). ASC reduction levels reached 82% of the total Cyt reduction obtained with ASC ⫹ dithionite, and the specific concentration of the ASC-reducible protein was 400 pmol mg⫺1 protein. DISCUSSION

Cyts b561 constitute a newly identified class of proteins in plants and animals involved in ASCmediated 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 ASCmediated 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; Verelst and Asard, 2003). One of the putative Cyts b561, CYBASC1, was expressed in yeast, and by spectroscopic characterization, we demonstrated its Cyt b nature and ASC reducibility. Moreover, the ␣-band absorbance characteristics are very similar to that observed for the mammalian chromaffin granule Cyt b561, with a characteristically asymmetrical peak and absorbance maximum near 561 nm (Tsubaki et al., 1997). Our results also indicate that inclusion of a C-terminal FLAG tag interferes with the expression of functionally active CYBASC1. 730

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; Horemans et al., 1994). However, it is not clear which of the four isoforms is represented by this activity. The presence of ASC-reducible Cyts b in other membranes than the PM has been suggested, but the location has not previously been identified (Asard et al., 1987; Scagliarini et al., 1998). To study the subcellular localization of the plant Cyts b561, we generated polyclonal C-terminal peptide antibodies against CYBASC1. The peptide antibodies specifically recognize CYBASC1, but not any of the other CYBASC isoforms, expressed in yeast. Moreover, the CYBASC1 antibodies crossreact with a partially purified ASC-reducible b-type Cyt from Arabidopsis PM fractions with a molecular mass similar to the predicted mass of CYBASC1. 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 TOenriched 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, TOenriched membrane fractions contain an ASCreducible b-type Cyt with an ␣-band wavelength

Figure 6. TO-enriched membrane fractions from Arabidopsis contain an ASC-reducible b-type Cyt. Reduced-minus-oxidized absorbance spectra demonstrate the presence of an ASC-reducible b-type Cyt in TO-enriched membrane fractions obtained from Arabidopsis leaves. The ␣-band is asymmetric and has a wavelength maximum near 561 nm. Spectra were recorded after oxidation by ferricyanide and subsequent reduction with ASC or ASC ⫹ dithionite, as described in “Materials and Methods.” Sample protein concentration ⫽ 1.03 mg mL⫺1. Plant Physiol. Vol. 134, 2004


maximum near 561 nm. Our results are also consistent with the presence of a b-type Cyt in TO-enriched membrane fractions obtained from soybean (Glycine max) hypocotyls (Barr et al., 1986). Four genes with highly conserved Cyt b561 properties have been identified in Arabidopsis (Asard et al., 2001; Verelst and Asard, 2003). Although the CYBASC1 antibodies specifically recognize this Cyt b561 isoform, it is possible that more than one Cyt b561 is located at the TO, and, consequently, that the spectroscopic measurements reflect the reduction by ASC of more than one Cyt b561. We observed that the CYBASC1 antibodies crossreact with a minor protein derived from a PMenriched 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; Chen et al., 2002; Be´ rczi and Asard, 2003; Be´ rczi et al., 2003). In fact, detection of marker proteins demonstrates the presence of TO membranes in the PM-enriched fraction. Therefore, the protein in the PM-enriched fraction recognized by the CYBASC1 antibodies is most likely derived from the TO. 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; Otegui et al., 2002), and recent observations point to the importance of redox reactions in this organelle. For example, redox agents control the activity of the plant vacuolar H-ATPase (Carpaneto et al., 1999; Tavakoli et al., 2001), and a phenol/peroxidase-based H2O2 scavenging mechanism has been identified in vacuoles (Yamasaki and Grace, 1998). Moreover, Cyt b5 reductases have been localized to the TO of maize (Zea mays) roots, but their function is still unknown (Bagnaresi et al., 2000). The presence of an ASCreducible Cyt b561 in the TO highlights the role of redox reactions in this organelle. TO Cyts b561 are likely to participate in ASCmediated 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) and little is known about its function or turnover. It has been suggested that cytoplasmic ASC diffuses freely into the vacuolar lumen (Rautenkranz et al., 1994), apparently arguing against a role in ASC regeneration for the TO Cyt b561. Mammalian Cyts b561 have also been sugPlant Physiol. Vol. 134, 2004

gested to function as iron reductases (McKie et al., 2001; Vargas et al., 2003). Vacuoles are possibly involved in cellular iron homeostasis, as is the case in yeast (Li et al., 2001). A putative metal transporter, AtNRAMP3, has recently been localized to the vacuolar membrane, and is suggested to mobilize vacuolar metal pools upon iron starvation (Pich et al., 2001; Yamaguchi et al., 2002; Thomine et al., 2003). In light of the possible iron reductase activity of Cyts b561, CYBASC1 may participate in reduction of vacuolar iron before transport into the cytosol. 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. MATERIALS AND METHODS 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 ␮mol m⫺2 ⫺1 s 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 (Be´ rczi et al., 2003). PM-depleted lower phase intracellular membranes (IMs) were collected, pelleted, and resuspended in buffer containing 10 mm Tris-HCl and 250 mm Suc, pH 7.5. Purified vacuolar membranes (TO) were a kind gift from Dr. Eduardo Blumwald (University of California, Davis) and were isolated as described (Blumwald and Poole, 1985). Proteins from the PM preparations were solubilized by treatment with 1% (w/v) nonaethylene glycol monododecyl ether (C12E9) and were separated by ionexchange chromatography (Mono-Q, FPLC; Amersham Pharmacia Biotech, Piscataway, NJ; Be´ rczi et al., 2003).

Membrane Fractionation by Suc Gradients 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.

Marker Enzyme Assays and Organelle-Specific Antibodies 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; Chanson et al., 1984). CCO activity was measured in the presence of 0.025% (v/v) Triton X-100 to permeabilize

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membranes and release latent activity. Chloroplast membranes were determined by measuring chlorophyll concentrations after extraction of 30 ␮L of the fraction in 1 mL of ethanol (95%, w/v). Total chlorophyll content was calculated using C(␮g mL⫺1) ⫽ 5.24 A664.2 ⫹ 22.2 A648.2 (Lichtenthaler, 1987; Hong et al., 1999). 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). Endoplasmic reticular membranes were detected with antibodies against a GTP-exchange protein (Sec12, 43 kD, 1:500; Bar-Peled and Raikhel, 1997; Secant Chemicals Inc., Winchendon, MA). Vacuolar membranes were detected with antibodies against the H⫹pumping pyrophosphatase (V-PPiase, 64.5–67 kD, 1:10,000; gift from Dr. Philip Rea, University of Pennsylvania, Philadelphia; Rea et al., 1992). Monoclonal antibodies against the ␤-ATPaseE subunit of the mitochondrial F0-F1 ATPase (55 kD, 1:10; gift from Dr. Tom Elthon, University of Nebraska, Lincoln; Luethy et al., 1993) were used as a marker for inner mitochondrial membranes. Antibodies against the Lewis a-containing N-glycan epitope of a Golgi-intrinsic protein (JIM84, 1:10; gift from Dr. Chris Hawes, Oxford Brooks University, Oxford, UK; Fitchette et al., 1999) were used to detect Golgi membranes. Protein concentrations were determined as described (Markwell et al., 1978) using bovine serum albumin as a standard. Data shown in Figure 4 are representative results from four independent experiments.

Cyt Determinations The concentration of b-type Cyts in Suc gradient fractions was determined from the reduced-minus-oxidized difference spectra (Asard et al., 1989). Absorbance spectra were recorded in split-beam mode (SLM-Aminco DW2000 spectrophotometer, SLM/Aminco, Urbana-Champaign, IL) at room temperature in 600-␮L samples in the absence or presence of ASC (10 mm) or ASC ⫹ dithionite (dithionite added as crystals) against the same amount of ferricyanide-oxidized Cyts. 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 reducedminus-oxidized difference spectra using the absorbance of the ␣-band maximum and a millimolar extinction coefficient of 20 mm⫺1 cm⫺1.

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). In addition to the primary antibodies described earlier, the M2 anti-FLAG monoclonal antibody (Sigma, St. Louis) was used to detect the recombinant proteins in yeast. Protein-antibody complexes were detected by horseradish peroxidase-conjugated secondary antibodies (enhanced chemiluminescence detection kit; Amersham Pharmacia Biotech). All results shown are representative blots from two to four independent repetitions.

Plasmid Constructs, Yeast Transformation, and Yeast Membrane Preparation 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) was used. Primers were designed to include EcoRI and SpeI sites for cloning into the pESC-His expression vector, downstream of the GAL10 Gal-inducible promoter and in-frame with a C-terminal sequence for the FLAG epitope. Sequences were confirmed by DNA sequencing at the University of Nebraska-Lincoln Genomic Core Research Facility. For transformation, yeast cells (strain YPH499, ura3-52 lys2-801amberade2101ochre trp1-⌬63 his3-⌬200 leu2-⌬1) were grown in synthetic dextrose (SD) minimal medium (Stratagene, La Jolla, CA) and transformation was performed according to the manufacturers instructions. Transformed lines were selected on SD dropout medium lacking His (SD-His). For the induction of protein expression, overnight cultures were grown in SD-His and were transferred to 500 mL of synthetic Gal minimal medium containing 2% (w/v) Gal. 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 ␮g 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.

Antibody Production and Purification 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.

Distribution of Materials Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.

ACKNOWLEDGMENTS 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.

Gel Electrophoresis and Western Blots

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Proteins in the membrane fractions were resolved by SDS-PAGE electrophoresis according to Laemmli (1970) using 12% (w/v) acrylamide gels (Figs. 2 and 4) or precast 4% to 20% acrylamide gradient gels (Bio-Rad, Hercules, CA; Figs. 1 and 3) and were transferred onto polyvinylidene difluoride membranes (Bio-Rad) with a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in 25 mm Tris, 182 mm Gly, and 0.1% (w/v) SDS.

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