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First published online December 4, 2003; 10.1104/pp.103.031484 Plant Physiology 134:182-193 (2004) © 2004 American Society of Plant Biologists Molecular and Cell Biology of a Family of Voltage-Dependent Anion Channel Porins in Lotus japonicus1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany (M.W., B.T., M.K.U.); Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, P.O. Box 1600, Canberra, Australian Capital Territory 2601, Australia (B.T.); and John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (N.B.)
Voltage-dependent anion channels (VDACs) are generally considered as the main pathway for metabolite transport across the mitochondrial outer membrane. Recent proteomic studies on isolated symbiosome membranes from legume nodules indicated that VDACs might also be involved in transport of nutrients between plants and rhizobia. In an attempt to substantiate this, we carried out a detailed molecular and cellular characterization of VDACs in Lotus japonicus and soybean (Glycine max). Database searches revealed at least five genes encoding putative VDACs in each of the legumes L. japonicus, Medicago truncatula, and soybean. We obtained and sequenced cDNA clones from L. japonicus encoding five full-length VDAC proteins (LjVDAC1.1–1.3, LjVDAC2.1, and LjVDAC3.1). Complementation of a yeast (Saccharomyces cerevisiae) mutant impaired in VDAC1, a porin of the mitochondrial outer membrane, showed that LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1, but not LjVDAC1.3, are functional and targeted to the mitochondrial outer membrane in yeast. Studies of the expression pattern of the five L. japonicus VDAC genes revealed largely constitutive expression of each throughout the plant, including nodules. Antibodies to LjVDAC1.1 of L. japonicus and the related POM36 protein of potato (Solanum tuberosum) recognized several proteins between 30 and 36 kD on western blots, including LjVDAC1.1, LjVDAC1.2, LjVDAC1.3, and LjVDAC2.1. Immunolocalization of VDACs in L. japonicus and soybean root nodules demonstrated their presence on not only mitochondria but also on numerous, small vesicles at the cell periphery. No evidence was found for the presence of VDACs on the symbiosome membrane. Nonetheless, the data indicate that VDACs may play more diverse roles in plants than suspected previously.
Porins are a diverse group of  -barrel proteins that fulfill a variety of functions in prokaryotes and eukaryotes. They are located in the outer membranes of Gram-negative (Delcour, 2002 ) and -positive bacteria (Riess et al., 1999, 2001; Lichtinger et al., 1998), mitochondria of eukaryotes (Benz, 1994), and plastids of plants (Fischer et al., 1994; for review, see Bolter and Soll, 2001). In addition to the common -barrel fold and perhaps related to this pore-forming structure, porins share the electrophysiological property of symmetric voltage gating (Bainbridge et al., 1998). Porins can adopt two conformational states: an open state, which is selective for small anions, and a closed state, in which conductivity decreases and selectivity for cations increases. Eukaryotic porins show voltage gating at potentials of ±20 to 40 mV, whereas an electrical potential of ±100 mV is needed to close certain bacterial porins (Benz, 1994). The physiological significance of voltage gating remains unclear, however, given that porins are generally located in "nonenergized" membranes (Blachly-Dyson and Forte, 2001).
Despite uncertainty about the physiological significance of voltage gating in porins, this feature has been used to name one important family of these proteins, the voltage-dependent anion channel (VDAC) family in eukaryotes. VDAC protein sequence and function have been conserved during evolution. Thus, homologs have been found in yeast (Saccharomyces cerevisiae), animals, and plants, and VDACs from animals (Blachly-Dyson et al., 1993
We were drawn to the study of VDACs in legumes after learning that they might be located in the peribacteroid or symbiosome membrane (SM) of pea (Pisum sativum) and Lotus japonicus nodules (Saalbach et al., 2002
Isolation and Sequence Analysis of Full-Length cDNAs Encoding Five Different VDACs in L. japonicus
Amino acid sequence data obtained from proteins of isolated SM from soybean (Glycine max; M. Wandrey, B. Trevaskis, and M.K. Udvardi, unpublished data), pea (Saalbach et al., 2002 The five L. japonicus VDACs encode proteins that are either 276 or 277 amino acids in length with calculated molecular masses in the range from 29 to 30 kD. They show between 44% to 88% sequence identity and form three main evolutionary branches (Fig. 1).
To identify conserved or variable domains that may be involved in secondary structure formation, targeting, or regulation of channel activity, deduced protein sequences of all five clones were aligned. In addition, secondary structure prediction using PROFsec (Rost, 1996
BLAST searches (Altschul et al., 1990 The phylogenetic relationships between VDACs from legumes and other plants are depicted in Figure 3. The dendrogram splits into five main branches, some of which contain monocot or dicot VDACs exclusively. Branch I contains only two plant VDACs, one from the single-celled organism Chlamydomonas reinhardtii and one from the legume M. truncatula. BLAST searches of these proteins revealed that they are more closely related to eukaryotic VDACs of fungi, mammals, and insects. Branch II contains VDACs from both dicotyledonous and monocotyledonous plants. Branches III and IV contain VDACs from dicots and monocots, respectively. Branch V is the largest branch with multiple VDAC isoforms of the dicots L. japonicus, soybean, M. truncatula, and potato (Solanum tuberosum). Some of these may have evolved by gene duplication after the monocot/dicot split, such as the L. japonicus VDACs LjVDAC1.1 and LjVDAC1.2, which exhibit high sequence identity (88%).
Division of labor between different proteins of the same family is often indicated by distinct patterns of gene expression in time and/or space within an organism. To determine whether specialization of L. japonicus VDAC function could be programmed at the level of transcription, we used RNA gel-blot analysis and real-time reverse transcriptase (RT)-PCR to monitor expression in different organs. Northernblot analysis was performed with total RNA from leaves, roots, flowers, and nodules of 8-week-old L. japonicus plants and showed constitutive expression of VDAC genes throughout the plant (data not shown). However, dot-blot analysis with in vitrotranscribed RNA revealed cross-hybridization among cloned LjVDAC genes. Therefore, we employed real-time RT-PCR to increase specificity and sensitivity of transcript detection. Leaf, root, and nodule poly(A+) RNA from 8-week-old plants was reverse transcribed and the resulting cDNA subjected to PCR analysis. Primers were designed to bind to the 3'-untranslated regions of LjVDAC1.1 to 1.3, LjVDAC2.1, and LjVDAC3.1 and tested for their specificity before use. Transcript levels of a polyubiquitin gene, which is expressed constitutively in L. japonicus (Colebatch et al., 2002
The functionality of LjVDAC1.1 to 1.3, LjVDAC2.1, and LjVDAC3.1 was assessed by complementation of yeast mutant strain HR1-2A (Dihanich et al., 1987
The analysis of VDAC expression in L. japonicus was extended to the protein level. Western blots were performed with affinity-purified anti-LjVDAC1.1 and anti-POM36 antibodies (Heins et al., 1994 The two antibodies were used to detect VDAC proteins in crude extracts and microsomal fractions from different organs of L. japonicus and soybean. Western-blot analysis using anti-LjVDAC1.1 antibody revealed two or three distinct VDAC bands around 32 to 36 kD in extracts of leaves, roots, and nodules (Fig. 5C). In L. japonicus extracts, these bands may correspond to LjVDAC1.1, LjVDAC1.2, and LjVDAC1.3. The anti-POM36 antibody recognized these and two additional bands at 30 and 38 kD (Fig. 5E). Both antibodies also cross-reacted with soybean VDACs. Specificity of the antibodies was tested using antibodies that were blocked with purified LjVDAC1.1 protein. Blocked anti-LjVDAC1.1 antibody detected no proteins on western blots of L. japonicus or soybean extracts (Fig. 5D), whereas blocked anti-POM36 antibody still cross-reacted with the 30- and 38-kD bands, which, therefore, might not be VDACs (Fig. 5F). The presence of VDACs in microsomal fractions of L. japonicus roots and nodules was also investigated. Anti-LjVDAC1.1 antibody detected only two protein bands in microsomal fractions from L. japonicus roots and nodules, instead of the three bands detected in total protein extracts (Fig. 5G). This indicated that one of the LjVDAC isoforms, possibly LjVDAC1.3, was located on a membrane that was removed by centrifugation during preparation of microsomal fractions.
The subcellular location of VDACs in L. japonicus and soybean nodules was investigated further using western blot and immunolocalization methods. SM was purified from soybean nodules via Percoll gradients (Panter et al., 2000 To establish more clearly the subcellular location of VDACs in L. japonicus and soybean nodules, we carried out immunofluorescence and immunogold labeling of tissue sections, using affinity-purified anti-LjVDAC1.1 antibody. Anti-LjVDAC1.1 antibody bound to punctate structures in the cell periphery close to, or associated with, the plasma membrane in L. japonicus and soybean nodules (Fig. 6, A and B). Control labeling with antibodies against the mitochondrial protein, Cyt c (Fig. 6C); the peroxisomal protein, uricase (Fig. 6D); and the plastidial protein, ferritin (Fig. 6E) were carried out on soybean nodule sections because of lack of cross-reactivity of some of the antibodies with the corresponding L. japonicus proteins. Peroxisomes and plastids were clearly larger (2–4 µm) than the structures labeled with anti-LjVDAC1.1. Thus, peroxisomes and plastids were not labeled by anti-LjVDAC1.1 antibody. An overlay of Cyt c labeling (Fig. 6F) and LjVDAC labeling (Fig. 6G) showed that a subset of LjVDAC-labeled structures were mitochondria (Fig. 6H, yellow). However, the anti-LjVDAC1.1 antibody also labeled vesicular, non-mitochondrial structures close to, or associated with, the plasma membrane that were not labeled with anti-Cyt c antibody (Fig. 6H, green).
Further characterization of VDAC-containing structures was done using immunogold labeling of L. japonicus nodule sections with the anti-LjVDAC1.1 antibody. Once again, two types of structures were labeled: abundant 200- to 500-nm vesicular structures near the plasma membrane (Fig. 7A) and less abundant but larger (typically >1 µm) mitochondria (Fig. 7B). Mitochondria exhibited internal cristae typical of this organelle, whereas the small vesicles did not.
Symbiosomes in nodule sections were not labeled with anti-LjVDAC1.1 antibody (Figs. 6 and 7), even though a VDAC antigen band was detected in SM preparations on western blots (Fig. 5). This apparent contradiction suggests that the SM preparations illustrated in Figure 5 were actually contaminated with another membrane type.
VDAC genes are present in fungi, animals, and plants. However, the numbers of homologs in different plant species exceed those in other eukaryotes. For example, Arabidopsis has five VDAC genes, whereas humans have three and yeast only two. At least five VDAC genes have been uncovered in each of L. japonicus, M. truncatula, and soybean by EST sequencing projects. Plant VDAC genes fall into five distinct subfamilies; the largest of these appear to have arisen since the divergence of monocots and dicots (Fig. 3). Three of the five L. japonicus VDAC genes characterized here fall into this category. Two of these, LjVDAC1.1 and LjVDAC1.2, are very similar in sequence and have a matching pair of genes in soybean, which may reflect a gene duplication event before the divergence of the Glycine and Lotus genera. Relatively recent gene duplication events in several other lineages are revealed by phylogenetic analysis (Fig. 3). Presumably, such events have provided the plant kingdom with an opportunity to diversify the physiological roles of VDAC proteins.
One way in which specialization of gene/protein function may come about during evolution is through a change in the pattern of gene expression. To determine whether any of the L. japonicus VDAC genes are expressed in an organ-specific manner, we carried out real-time RT-PCR analysis of leaves, roots, and nodules from mature L. japonicus plants. All five L. japonicus VDAC genes were expressed at high levels, although there were slight differences in the level of expression between genes and organs. Differential expression of VDAC genes during development has been reported for wheat (Triticum aestivum) and rice (Oryza sativa; Elkeles et al., 1995
Protein specialization can arise from changes in protein sequence and structure, which can alter the biochemical properties of a protein and/or its sub-cellular location. Clear differences in primary structure were revealed by comparisons of the deduced amino acid sequences of the five L. japonicus VDACs (Fig. 2). However, the predicted secondary structural elements, including an
The N-terminal Functionality and intracellular location of L. japonicus VDAC proteins was examined by expressing them in a VDAC1-deficient yeast mutant. Complementation or suppression of the por- mutation in yeast strain HR1-2A by LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1 demonstrated not only that these proteins function in yeast but also that they are targeted to the OMM (Fig. 4). This is the first time, to our knowledge, that plant VDACs have been shown to function in yeast, which opens up the possibility to carry out more detailed biochemical characterization of these proteins in the future. LjVDAC1.3 was unable to complement the yeast mutant, although the protein was expressed in transformed cells (Fig. 5, A and B). Immunolocalization showed that LjVDAC1.3 was not targeted to the OMM in yeast (data not shown), which provides an explanation for its inability to complement the mutant. Interestingly, LjVDAC1.3 is the least related of all the L. japonicus VDACs to animal and yeast VDACs. Sequence differences must account for the altered intracellular targeting of this protein in yeast, and this result raises the possibility that the protein is not located on the OMM in L. japonicus.
The anti-LjVDAC1.1 antibody that we produced is highly specific for VDACs. It binds strongly to LjVDAC1.1, LjVDAC1.2, and LjVDAC1.3, weakly to LjVDAC2.1, and not at all to LjVDAC3.1 (Fig. 5A) or other L. japonicus proteins (Fig. 5, C and D). The anti-LjVDAC1.1 antibody also recognizes VDAC proteins in soybean (Fig. 5, C and D). In contrast, the anti-POM36 antibody cross-reacted strongly with two proteins in L. japonicus that could not be confirmed as VDACs (Fig. 5, E and F). Localization studies, using affinity-purified LjVDAC1.1 antibody together with Cyt c antibody, confirmed the mitochondrial location of some VDACs in root nodules (Fig. 6, F–H). However, anti-LjVDAC1.1 antibody also labeled distinct vesicular structures close to the plasma membrane, which were not labeled with anti-Cyt c antibody (Fig. 6, F–H). Because the anti-LjVDAC1.1 antibody cross-reacts with LjVDAC1.1 to 1.3, and LjVDAC1.1 and LjVDAC1.2 are targeted to the OMM in yeast, these proteins are likely to be located in the OMM in root nodules as well. In contrast, the LjVDAC1.3 protein, which was not targeted to the OMM of yeast and was probably absent from microsomal fractions (Fig. 5G), may be located in the non-mitochondrial, vesicular structures observed in root nodules. These structures were apparently not peroxisomes or plastids (Fig. 6). We can only speculate on the identity of these vesicles at this stage. VDAC proteins have been found in extra-mitochondrial membranes in animals, in the caveolea, or Tritoninsoluble fraction domains of plasma membranes (Bathori et al., 1999
Results from proteomics studies have indicated the presence of VDACs in SM preparations from pea and L. japonicus nodules (Saalbach et al., 2002 To summarize, we have characterized five VDAC porins from the model legume, L. japonicus, four of which are functional at the OMM when expressed in yeast. Transcripts of all five genes accumulate to similar, high levels throughout the plant. Immunolocalization of VDACs in L. japonicus and soybean root nodules revealed their presence not only on mitochondria but also on distinct, small vesicles, which remain to be identified. Our results indicate that the biological roles of VDAC proteins have diversified during the evolution of plants.
Plant Culture Lotus japonicus GIFU B-129-S9 and soybean (Glycine max cv Stevens) plants were grown in quartz sand-filled pots in a greenhouse under artificial light in a 16-h-light/8-h-dark rhythm. Inoculation of L. japonicus with Mesorhizobium loti strain R7A and of soybean with Bradyrhizobium japonicum USDA110 was done at the time of sowing and repeated 4 to 7 d later.
DNA manipulations such as plasmid purification, restriction digests, agarose gel electrophoresis, ligations, and screening of cDNA libraries were performed using standard protocols (Sambrook et al., 1989
Leaves, stems, roots, flowers, and nodules from 6-week-old L. japonicus plants were harvested and ground to a fine powder in liquid nitrogen, using mortar and pestle, and RNA was extracted using the detergent-based method of Jacobsen-Lyon et al. (1995
Poly(A+) RNA was purified from 10 µg of DNase-treated total RNA, using the Qiagen Oligotex mRNA kit (Qiagen, Hilden, Germany). Approximately 10 ng of mRNA was then reverse transcribed with Superscript II RT (Invitrogen, Karlsruhe, Germany) using an oligo(dT) primer, to generate 50 µL of first strand cDNA. Real-time RT-PCR was performed using 1 µL of a 1/10 (v/v) dilution of the first strand cDNA reaction and the SYBRGreen reagent (Eurogentec, Seraing, Belgium) in a reaction volume of 25 µL on a GeneAmp 5700 Sequence Detection System (PE-Applied Biosystems, Foster City, CA). Primers were designed to the 3'-untranslated region of LjVDAC1.1 (forward, 5'-GATAATTT TGATCCTTGGCAAGAC-3'; and reverse, 5'-GAACACTTCTTAGCCAAGAAGAG-3'), LjVDAC1.2 (forward, 5'-ATGA TTTTGAATTGATAGGCTGCG-3'; and reverse, 5'-ACCCAGC AAGAAATTAACAGCTC-3'), LjVDAC1.3 (forward, 5'-TTTTGAGCCATTATGGCAAGA GC-3'; and reverse, 5'-AACACTT ATGTCCAATAGAACCAG-3'), LjPor2.1 (forward, 5'-TTG AAAGGAGGGCAGAATAA TTAG-3'; and reverse, 5'-GTCAAACAGAAGCCATTGGTGAT-3'), and LjPor3.1 (forward, 5'-GCCTTGTCATAAAGGTAAAACCAA-3'; and reverse, 5'-CTCTAGCCATTATGTAAGTATT TC-3'). Each primer pair amplified a single product, as shown by the melting temperature of the amplicons and agarose gel electrophoresis. Likewise, ubiquitin primers (forward, 5'-TTCACCTTGTGCTCCGTCTTC-3'; and reverse, 5'-AACAACAGCACACACAGACAATCC-3') were designed against the L. japonicus polyubiquitin gene (GenBank accession no. AW720576). Samples without template were used as negative controls. Expression data were normalized to ubiquitin and then compared using the formulae:
 CT is the difference in normalized CT values in tissues x and y, and Ration is the ratio of gene transcript level in tissue x compared with y.
Yeast strains HR1-2B (por+) and HR1-2A (por-) were a kind gift of Dr. Carla Koehler (University of California, Los Angeles; Dihanich et al., 1987
All protein extraction methods were carried out at 4°C, unless otherwise stated. Total protein extracts from L. japonicus and soybean organs were prepared from 8-week-old plants by harvesting fresh tissue into liquid nitrogen and grinding the frozen tissue with mortar and pestle to a fine powder. The powder was resuspended in an equal volume of extraction buffer (10 mM Tris [pH 7.5], 140 mM NaCl, 5 mM EDTA, 1% [v/v] Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Cell debris and insoluble components were pelleted twice by centrifugation at 14,000 rpm for 10 min. Protein concentration of supernatants was determined according to Bradford using the Bio-Rad Protein Assay Kit I (Bio-Rad, Munich). Approximately 20 µg of protein was loaded per lane.
SMs from soybean root nodules of 8-week-old plants were prepared as described by Panter et al. (2000 Crude yeast protein extracts were prepared by first pelleting 5 mL of cells of an overnight culture and resuspending them in 150 µL of LDS-Sample buffer (Invitrogen). After heating for 10 min at 95°C, a half volume of glass beads was added, and the cells were vortexed for 10 min before being subjected to SDS-PAGE. Approximately 20 µg of protein, in 10 µL of extract, was loaded per lane. To obtain polyclonal anti-LjVDAC1.1 antiserum, the open reading frame of the LjVDAC1.1 cDNA was amplified with primers containing either BamHI or HindIII restriction sites (forward, 5'-GGATCCATGGCTAAGGGTCCTGGTCTC-3'; and reverse, 5'-AAGCTTCC CAATGTCTTGCCAAGGATC-3'). After subcloning the 871-bp PCR fragment into pGEMTEasy (Promega, Mannheim, Germany), the BamHI-HindIII fragment was isolated and ligated to a poly-His-encoding sequence in vector pQE30 (Qiagen). Protein expression in Escherichia coli and protein purification using nickel affinity columns was performed as per the manufacturer's instructions (QIAexpressionist, Qiagen). Purified protein was then injected into rabbits (Pineda Antikörper Service, Berlin), and polyclonal antisera were collected 61 and 90 d after immunization. Antiserum was then affinity purified using Sepharose columns with immobilized LjVDAC1.1 protein (Pineda Antikörper Service).
After separation on NuPage precast gels (Invitrogen), proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Ein-beck, Germany) overnight at 70 mA or for 150 min at 400 mA with an XCell II Blot Module (Invitrogen) in 25 mM Tris-Base, 190 mM Gly, and 20% (v/v) methanol. Protein detection on western blots using 1:1,000 (v/v) diluted primary antibody, alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:1,500 [v/v] dilution, Promega), and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate tablets (Roche Diagnostics, Mannheim, Germany) was performed as described by Blake et al. (1984
Root nodules of around 8-week-old L. japonicus and soybean plants were fixed overnight in PEM (50 mM 1,4-Piperazinediethanesulfonic acid (PIPES) [pH 6.8], 5 mM EDTA, and 5 mM MgSO4), containing 3.7% (w/v) paraformaldehyde, under a slight vacuum. Using a VT1000S Vibratome (Leica, Bensheim, Germany), 100-µm sections were taken and mounted onto poly-L-Lys-coated slides (Poly-Prep-Slides, Sigma). Sections were treated for 10 min with 0.5% (w/v) Cellulase R10 in PEM (Yakult, Tokyo) before blocking with PEM, containing 3% (w/v) BSA, 0.1% (v/v) Tween20, and 20 mM Lys for 2 h at room temperature. Affinity-purified anti-LjVDAC1.1 and anti-Cyt c antibodies (BD Biosciences, Heidelberg) were applied in a 1:10 (v/v) dilution, anti-uricase antibody (VandenBosch and Newcomb, 1986
Root nodule pieces from 8-week-old plants were fixed overnight at 4°C in 0.1 M sodium phosphate buffer (pH 7.3), containing 4% (w/v) paraformaldehyde. Samples were then dehydrated for 6 h in 30% (v/v) ethanol, 3 h in 50% (v/v) ethanol at room temperature, and overnight in 70% (v/v) ethanol at 4°C. Afterward samples were embedded in LR-White (LRW) Resin Medium Grade (Plano, Wetzlar, Germany) by infiltration for 6 h in a 1:1 (v/v) LRW:ethanol mixture and for 24 h in pure LRW at 4°C. Polymerization was carried out for 2 d at 50°C. Ultrathin sections (90 nm) were taken with an Ultracat E ultramicrotome (Leica) and mounted onto pyroxylin- and carboncoated 200-mesh gold grids (Plano). For immunogold staining, the sections were blocked for 1 h at RT in phosphate-buffered saline (PBS; 10 mM sodium phosphate buffer [pH 7.5] and 130 mM NaCl), containing 1% (w/v) Aurion-BSA (Science-Services, Munich) and 0.1% (v/v) Tween 20. Affinity-purified LjVDAC1.1 antibody was diluted 1:10 (v/v) in incubation solution (PBS, 0.1% [w/v] Aurion-BSA, and 0.01% [v/v] Tween20) and applied overnight at 4°C. After three washes for 15 min in wash buffer (PBS and 0.1% [v/v] Tween20), the gold-conjugated secondary antibody (10 nm of gold, British BioCell International, Cardiff, UK) diluted 1:30 (v/v) in incubation solution was applied for 3 to 4 h at room temperature. After washing three times for 15 min in (successively) wash buffer, PBS, and distilled water, the grids were contrast stained with uranyl acetate and lead citrate. The labeled sections were monitored with a Joel Transmission electron microscope (Tokyo, Japan) at a voltage of 80 mV and photographed.
We would like to thank the Kazusa DNA Research Institute (Chiba, Japan) for VDAC cDNA clones and Drs. Carla Koehler (University of California, Los Angeles), Udo Schmitz (Universität Hannover, Germany), Jean-Francois Briat (University of Montpellier, France), and Desh Pal Verma (Ohio State University, Columbus) for the VDAC1-deficient yeast mutant and antibodies to yeast VDAC1, POM36, ferritin, and uricase, respectively. We also thank Kim Findlay (John Innes Centre, Norwich, UK) for help with electron microscopy. Received August 7, 2003; returned for revision September 11, 2003; accepted September 26, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.031484.
1 This work was supported by the Deutsche Forschungsgemeinschaft and by the Max Planck Society. * Corresponding author; e-mail udvardi{at}mpimp-golm.mpg.de; fax 49–331–567–8250.
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-helical region (boxed H). The positions of 
