| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online November 20, 2003; 10.1105/tpc.017541 American Society of Plant Biologists Poplar Metal Tolerance Protein 1 Confers Zinc Tolerance and Is an Oligomeric Vacuolar Zinc Transporter with an Essential Leucine Zipper Motif
a Biology Department, University of York, York YO10 5YW, United Kingdom 2 To whom correspondence should be addressed. E-mail michel.chalot{at}scbiol.uhp-nancy.fr; fax 33-3-83-6842-92
Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. Here, we describe the identification, characterization, and localization of PtdMTP1, a member of the CDF family from the hybrid poplar Populus trichocarpa x Populus deltoides. PtdMTP1 is expressed constitutively and ubiquitously, although at low levels. Heterologous expression in yeast showed that PtdMTP1 was able to complement the hypersensitivity of mutant strains to Zn but not to other metals, including Cd, Co, Mn, and Ni. PtdMTP1 fused to green fluorescent protein localized to the vacuolar membrane both in yeast and in plant cells, consistent with a function of PtdMTP1 in zinc sequestration. Overexpression of PtdMTP1 in Arabidopsis confers Zn tolerance. We show that PtdMTP1, when expressed in yeast and Arabidopsis, forms homooligomers, a novel feature of CDF members. Oligomer formation is disrupted by reducing agents, indicating possible disulfide bridge formation. PtdMTP1 also contains a conserved Leu zipper motif. Although not necessary for oligomer formation, Leu residues within this motif are required for PtdMTP1 functional activity.
Transition elements such as Fe, Co, Ni, Cu, and Zn play a wide variety of roles in biology as enzyme cofactors and must be absorbed from the soil by plants. However, either naturally or as a result of human activity, these metals can be present at potentially toxic concentrations. A number of other metal ions with no known biological function—including Cd, Pb, and Hg—also are potentially highly toxic for plants. Two basic strategies for decreasing the toxicity of metals are apparent: chelation and efflux from the cytosol, either into the apoplast or by intracellular sequestration. A number of cation transporter families have emerged recently and been classified in plants (Mäser et al., 2001  ), and although it is apparent that some (e.g., the Zrt-, Irt-like protein [ZIP] family) are involved in the high-affinity uptake of metals for nutritional purposes, overexpression of a member of another family (the cation diffusion facilitator [CDF] family) can lead to decreased metal toxicity and enhanced accumulation (van der Zaal et al., 1999).
Interest regarding transporters that facilitate the accumulation of potentially toxic metals has centered on their potential exploitation in phytoremediation. Phytoremediation is an emerging technology potentially effective and applicable to a number of different contaminants and site conditions (Lasat, 2002
Among essential metals, zinc plays critical roles in a wide variety of biochemical processes; therefore, intracellular zinc concentrations must be maintained at adequate levels to support cell growth (Gaither and Eide, 2001
Here, we report the identification of poplar MTP1 (PtdMTP1), which encodes a protein with features typical of CDF proteins. PtdMTP1 was identified in a poplar EST database (Kohler et al., 2003
Molecular Analysis of a Poplar Zinc Transporter To examine the molecular basis of Zn homeostasis in poplar, we searched the EST database of Populus trichocarpa x Populus deltoides roots (Kohler et al., 2003 ) for sequence homology with known Zn transporters of the ZAT/MTP/CDF family. We identified a cDNA homologous with plant and yeast zinc transporters. We named the poplar homolog PtdMTP1 (Populus trichocarpa x deltoides metal tolerance protein), in accord with the suggestion in a recent review (Mäser et al., 2001) and the new nomenclature given by Delhaize et al. (2003). The PtdMTP1 cDNA corresponds to a 393-residue protein (43.5 kD). Analysis of predicted sequence from the cDNA showed 73.7% identity with Arabidopsis ZAT (now known as AtMTP1; Mäser et al., 2001). The hydropathy profile of PtdMTP1, generated with the Kyte and Doolittle (1982) algorithm (http://www.expasy.org/cgi-bin/protscale.pl), predicts six hydrophobic domains of sufficient length to be considered potential membrane-spanning domains, with a long loop separating transmembrane domains IV and V (data not shown). PtdMTP1 possesses the most conserved region of the CDF family proteins, which encompasses transmembrane spans I to IV. Within this region, the CDF family–specific signature sequence (Paulsen and Saier, 1997), which begins with the fully conserved Ser-80 and continues just past the fully conserved Asp-93, was identified. A hydrophilic, His-rich stretch, to which zinc probably binds (Bloss et al., 2002), extends from His-182 to His-232.
A phylogenetic tree for the CDF family proteins is shown in Figure 1. This tree is a revised and extended version of that presented by Mäser et al. (2001)
PtdMTP1 Is Expressed Constitutively Reverse transcription (RT)–PCR analyses demonstrated that PtdMTP1 was expressed constitutively in most poplar tissues, as shown in Figure 2A. The highest level of PtdMTP1 transcript was detected in mature leaves and roots. We further measured the expression of PtdMTP1 by RT-PCR in root and leaf from poplar seedlings grown axenically and found that transcript level was not affected by exposure to low or high concentrations of Zn (0.038, 0.38, and 3.8 mM) for short or extended periods (3 to 5 days) (data not shown). The additional metals Mn, Cd, and Ni also were checked, and we found no response to these either. Transcript profiling experiments to study poplar adventitious root development revealed that PtdMTP1 was expressed constitutively, although at low levels, as shown in Figure 2B. Expression was independent of root development stage, in contrast to that of the extensin gene EXT1. The low expression level also was reflected by the low abundance of the PtdMTP1 clone in a Populus trichocarpa x deltoides root cDNA library (just 1 in >7000 ESTs [Kohler et al., 2003 ]; http://mycor.nancy.inra.fr/poplardb/index.html) and in the Populus spp.cDNA libraries (8 in >95,151 ESTs from various tissues; http://poppel.fysbot.umu.se). Fortuitously, in other cDNA array projects performed to study drought stress, root colonization by the ectomycorrhizal fungus Paxillus involutus, and leaf infection by the rust fungus Melampsora larici-populina, we found that PtdMTP1 expression was not affected significantly (data not shown).
Expression of PtdMTP1 in Yeast Confers Tolerance to Zn but Not to Cd, Co, Mn, or Ni To characterize PtdMTP1 further, PtdMTP1 cDNA was expressed in S. cerevisiae mutant strains that are unable to grow in high concentrations of various metals, and growth was monitored on either control or metal-supplemented medium. Figure 3 shows that the wild-type strain was able to grow at high Zn concentrations (Figure 3B), whereas deletion of the ZRC1 gene, which encodes a transporter that sequesters Zn into the vacuole, rendered the mutant highly sensitive to Zn (Kamizono et al., 1989 ; Li and Kaplan, 1998). The Zn-sensitive phenotype of the zrc1 mutant was fully complemented by PtdMTP1. cot1 mutants are hypersensitive to both Zn and Co, because COT1 mediates the efflux of both ions into the vacuole (Conklin et al., 1992; Li and Kaplan, 1998; Lyons et al., 2000). Interestingly, although PtdMTP1 complemented the cot1 mutant phenotype when grown on Zn (Figures 3C and 3D), PtdMTP1 failed to complement when grown on Co (Figures 3E and 3F). We further transformed the ycf1, pmr1, and smf1 mutant strains, which are unable to grow on Cd, Mn, and Ni, respectively. YCF1 is an ABC transporter that confers Cd tolerance through the transport of Cd conjugates into the vacuole (Szczypka et al., 1994; Li et al., 1997). PMR1 is the yeast secretory pathway pump responsible for high-affinity transport of Mn2+ and Ca2+ into the Golgi and confers Mn tolerance by effectively removing Mn from the cytoplasm (Ton et al., 2002). SMF1 functions in the cellular accumulation of Mn, and the smf1 mutant was shown to be Ni sensitive (Supek et al., 1996). Transformation with PtdMTP1 did not restore the growth of ycf1 on 0.15 mM Cd (Figures 3G and 3H), of pmr1 on 3 mM Mn (Figures 3I and 3J), or of smf1 on 1.5 mM Ni (Figures 3K and 3L). The data obtained with the five mutant strains suggest that PtdMTP1 specifically transports Zn but not Co, Cd, Mn, or Ni.
PtdMTP1 Partially Rescues the Growth of Yeast Vacuolar Acidification Mutants under Zn Stress Yeast vacuolar acidification mutants are unable to acidify their vacuoles and have increased sensitivity to some heavy metals, possibly because proton-driven antiport on the vacuolar membrane is required for the sequestration of metals into the lumen (Ramsay and Gadd, 1997 ). Therefore, to gain insight into the dependence of PtdMTP1 on a proton gradient, two vacuole acidification mutants (vma8 and vph2) were transformed with PtdMTP1 and colonies were assayed subsequently for growth at different Zn concentrations. The VMA8 gene encodes a subunit of the catalytic domain of the vacuolar-type H+-ATPase, and the VPH2 protein is required for the biogenesis of a functional vacuolar ATPase (Bachhawat et al., 1993; Graham et al., 1995). Figure 4 shows that vma8 and vph2 are highly sensitive to Zn (no growth at 0.25 mM Zn) compared with the wild type (growth up to 15 mM; Figure 3). Interestingly, the growth of both acidification mutants was partially rescued when cells were transformed with PtdMTP1. These PtdMTP1-expressing transformants were able to grow at 0.25 mM Zn (Figure 4) and also partially at 0.5 mM Zn (data not shown).
PtdMTP1:GFP Is Targeted to the Vacuolar Membrane in Yeast Cells Phenotypic suppression by PtdMTP1 of the zrc1 phenotype indicates a putative vacuolar membrane localization of the transporter in yeast cells. We constructed the PtdMTP1:GFP fusion protein to determine the intracellular localization of PtdMTP1 more precisely. Because the PtdMTP1:GFP fusion gene complemented the zinc sensitivity of the zrc1 mutant (data not shown), the fusion protein was confirmed to retain its original function. The bright-field image in Figure 5A shows the clear presence of vacuoles in zrc1 yeast cells. At the standard image acquisition settings used for GFP visualization, autofluorescence from cells transformed with the untagged PtdMTP1 was absent (data not shown), so all detectable fluorescence in the transformants was GFP specific. Figure 5B shows that, when exponentially growing cells were analyzed by confocal laser scanning microscopy, green fluorescence resulting from PtdMTP1:GFP showed a ring-like pattern in the cells. We speculated that this ring-like pattern might reflect vacuolar membrane location. We then stained the vacuolar membrane with the lipophilic dye FM4-64 to identify the location of this organelle (Vida and Emr, 1995). After 60 min of internalization, FM4-64 was found exclusively at the vacuolar membrane in transformed S. cerevisiae cells (Figure 5C). Figure 5D shows that superposition of the GFP and FM4-64 images yields a coincident staining pattern, demonstrating that PtdMTP1 was localized to the vacuolar membrane. When the zrc1 mutant was transformed with the vector carrying only the GFP, the fluorescence was found throughout the cytosol (Figure 5F) and was not coincident with the staining of the vacuoles (Figures 5G and 5H).
PtdMTP1 Localizes to the Vacuolar Membrane of Plant Cells Prediction by TargetP (Emanuelsson et al., 2000 ) indicated no preferred location of PtdMTP1. Therefore, we tested experimentally whether the vacuolar location of the transporter in yeast cells reflects the location in the plant. The PtdMTP1:GFP fusion protein was expressed transiently in living onion epidermal cells (Scott et al., 1999), which then were subjected to analysis by confocal laser scanning microscopy. The bright-field image in Figure 6A shows that, although onion epidermal cells have a large vacuole that effectively contours the cell, a clear separation from the cell periphery was seen in the region of the nucleus (inset). The corresponding fluorescence image in Figure 6B shows a clear localization of GFP to the indented region, rather than the cell periphery, unambiguously demonstrating a vacuolar location. Membrane-lined transvacuolar strands of cytoplasm spanning the cell also produced fluorescence, another typical feature of a vacuolar membrane location (data not shown).
Transgenic lines of Arabidopsis expressing PtdMTP1:GFP were used to confirm the vacuolar location of PtdMTP1 found by transient expression in onion cells. In elongated root cells, the large central vacuole usually occupies most of the cell volume, and the vacuolar and plasma membranes are closely juxtaposed. Conversely, cells in the root tip region contain several small immature vacuoles, so we focused on the examination of GFP fluorescence in root tips. Figure 6C shows the presence of numerous intracellular globular localization patterns in those cells, strongly suggesting a vacuolar location. The defined peripheral location of the AtMGT1:GFP fusion protein, as described previously by Li et al. (2001) , is shown in Figure 6D as a control for plasma membrane localization.
PtdMTP1 Confers Zn2+ Tolerance in Planta
PtdMTP1 Forms Oligomers That Are Sensitive to Reducing Agents Size estimation by SDS-PAGE using metal blots indicated that AtZAT might form dimers when present in membranes (Bloss et al., 2002 ). To address whether PtdMTP1 forms oligomers, we used protein gel blot analysis. We expressed the PtdMTP1:GFP fusion protein in yeast cells and solubilized the membrane proteins with n-dodecyl  -D-maltoside. The total molecular mass of the fusion protein was 73 kD. Figure 8A shows the presence of two bands of  60 and 140 kD on protein gel blots when using GFP antibodies, with a third band running at 220 kD. Although these data imply that a substantial fraction of PtdMTP1, when expressed in yeast cells, exists in the form of dimers, it remained possible that PtdMTP1 forms heterodimers with an unknown protein of similar size and thus migrated as an apparent homodimer. To test this possibility, we generated a PtdMTP1:V5 fusion protein (49.5 kD) and performed similar nonreducing SDS-PAGE experiments. As shown in Figure 8B, PtdMTP1:V5 migrated as two bands of 50 and 100 kD, suggesting the presence of both monomeric and dimeric forms and consequently refuting the heterodimeric formation of PtdMTP1:GFP with another unknown protein of 80 kD. A third band running at 150 kD also was observed (Figure 8B). Figure 8C demonstrates that oligomer formation also occurred in planta. Therefore, whether expressed in planta or in yeast, PtdMTP1 proteins are likely to be present at least as dimers; nevertheless, the presence of higher molecular mass complexes cannot be excluded and are indicated in Figure 8.
To confirm the presence of oligomers, cotransformation of yeast cells with both PtdMTP1:V5 and PtdMTP1:GFP constructs was performed. Because the two tagged species have significantly different molecular masses, heterodimeric and homodimeric chimeric proteins are expected to migrate differentially on gels of low acrylamide concentration. In one approach, anti-GFP antibodies were used to detect PtdMTP1 proteins. Figure 9A shows that in addition to the homodimeric species running with an apparent molecular mass of 140 kD, a band with a lower mass of 120 kD also was detected, indicating the presence of heterodimeric PtdMTP1:V5/PtdMTP1:GFP complexes. Complementary data were obtained when the converse experiment was performed; that is, anti-V5 antibodies revealed the presence of a heterodimeric complex, which although appearing fainter, had a higher molecular mass compared with that of the homodimeric complex. We also considered the possibility that PtdMTP1 complexes could be formed during the extraction procedure. To test this possibility, we mixed cells that expressed either PtdMTP1:GFP alone or PtdMTP1:V5 alone and then processed the mixture for protein gel blot analysis as described above. Heterodimers were not observed when the blot was probed with anti-GFP antibodies or with anti-V5 antibodies (Figure 9A). To determine whether PtdMTP1 dimerization occurred through disulfide bridges, extracts containing the PtdMTP1:GFP dimers were treated with the reducing agent DTT before gel loading. As shown in Figure 9B, increasing DTT concentration in the sample led to increasing dissociation of the dimers. At 20 mM DTT, almost complete dissociation was observed. Similar results were found when extracts containing PtdMTP1:V5 were used as well as with the reducing agents -mercaptoethanol and tributylphosphine (data not shown).
PtdMTP1 Contains a Leu Zipper Motif That Is Necessary for Function The discovery that PtdMTP1 forms multimers possibly stabilized by S-S bridges encouraged us to examine the possibility that other motifs are involved in functional aspects of protein–protein interaction. The Scanprosite interface (www.expasy.ch/cgi-bin/scanprosite) enabled the identification of a Leu zipper (LZ) motif in the C-terminal PtdMTP1 sequence from residues Leu-293 to Leu-314. Figure 10 shows that this LZ domain is conserved among plant CDF proteins. The most notable feature of the LZ is the presence of a repeating pattern of Leu residues at every seventh position (LX6)n. More generally, however, the overall architecture of coiled coils is defined by a 4,3 hydrophobic repeat in the primary amino acid sequence. Thus, the hydrophobic positions a and d of the sequence (abcdefg)n fall on the same face of an  -helix. In some cases, the Leu residues are replaced by Ile or Val and can include "skips" and "hiccups" (Leung and Lassam, 1998). This explains the unsuccessful searches for LZ motifs in some other CDF proteins (e.g., AtMTP1) with search programs that are designed to detect only the conventional (LX6)n motif, even though the motifs are effectively present (Figure 10).
To determine the role of the conserved LZ in the oligomeric structure and biological activity of PtdMTP1:GFP, the heptadic Leu residues 293, 300, 307, and 314 were changed individually or in pairwise combination to Ala residues. Ala was chosen as a substituting residue for Leu because (1) Ala has a strong propensity to form an -helix and its substitution has minimal disruptive effects on the -helical structure characteristic of a LZ domain in proteins, and (2) Ala lacks the bulky hydrophobic side chain of Leu, which is believed to be the major hydrophobic force for a LZ domain to facilitate protein interaction (Luo et al., 1999). Figure 11 (bottom gel) shows that all of the mutant proteins were expressed at levels identical to that of the wild-type protein. Substitution of one or two of the four Leu residues did not alter the size of the fusion protein oligomer, as determined by gel electrophoresis, nor was the subcellular localization of the mutant PtdMTP1:GFP proteins changed. However, as shown in Figure 11, although the L293A substitution had no apparent effect on function, the L314A substitution completely abolished the functional complementation of the zrc1 mutant by PtdMTP1 on 15 mM Zn. Other single substitutions (L300A and L307A) showed intermediate patterns: the reduction in yeast growth at high Zn concentration was marked but not fully abolished. Double Leu substitution mutants confirmed the patterns observed with the single Leu mutants, with a slight additional effect in growth inhibition, especially for the L300A-L307A mutant (Figure 11).
Residue Substitutions in the CDF Motif Abolish Yeast Complementation PtdMTP1 contains the CDF signature, which begins with Ser-80 and continues just past the fully conserved Asp-93. We created four mutants (D86A, H89A, H89K, and D93A) by site-directed mutagenesis of either semi (D86 and H89) or fully (D93) conserved residues. All of the mutant proteins were expressed at levels identical to that of the wild-type protein (Figure 11, bottom gel), still formed dimers, and were localized on the vacuolar membrane (data not shown). However, as shown in Figure 11, all four mutants failed to complement the Zn hypersensitivity of zrc1 cells, as shown by the complete abolition of yeast growth on 15 mM Zn.
The novel transporter from the hybrid poplar Populus trichocarpa x deltoides, PtdMTP1, is a member of the CDF family. The CDF family can be subdivided into three (van der Zaal et al., 1999 ; Gaither and Eide, 2001) or four (Mäser et al., 2001) groups. Following the classification given by Gaither and Eide (2001), PtdMTP1 is most closely related to group-III CDF members (Figure 1). Heterologous expression of PtdMTP1 in yeast lacking either of the vacuolar cytoplasmic efflux carriers ZRC1 or COT1 complements only the zinc sensitivity of these yeast strains, strongly suggesting that PtdMTP1 is able to selectively transport zinc ions into the yeast vacuole. These properties reflect those of AtZAT (AtMTP1), which when reconstituted in proteoliposomes was shown to transport Zn but not Co (Bloss et al., 2002). However, the AtZAT gene did not rescue the zinc sensitivity of a zrc1 single mutant or a zrc1 cot1 double mutant strain of S. cerevisiae, but it did rescue that of a zrc1 mutant strain of S. pombe (Bloss et al., 2002).
A critical question in establishing the physiological function of CDF transporters relates to their cellular location. Table 1 shows for a number of CDF family members a range of disparate locations, including on most intracellular membranes and the plasma membrane. Thus, ShMTP1, a S. hamata CDF member that specifically transports Mn, was found recently to be targeted to the vacuolar membrane in planta but localized to endoplasmic reticulum membranes when expressed in yeast (Delhaize et al., 2003
In eukaryotes, two families of transporters have been implicated in zinc transport. ZIP family members play prominent roles in cytosolic zinc import, transporting zinc from outside the cell (Pence et al., 2000 ) or from within an intracellular compartment (Gaither and Eide, 2001). The second group of transporters, the CDF family, transports zinc in a direction opposite that of the ZIP transporters, promoting cytosolic zinc efflux. Chardonnens et al. (1999) reported evidence that transport across the vacuolar membrane plays an essential role in naturally selected zinc-tolerant Silene vulgaris. van der Zaal et al. (1999) suggested that increased Zn transport into the vacuoles of Zn-tolerant species such as S. vulgaris might be attributable to a ZAT-like protein, although data on ZAT localization were lacking. The present study demonstrates that the ZAT homolog PtdMTP1 is located at the vacuolar membrane, which is consistent with a role for PtdMTP1 in zinc sequestration. Zinc transporters—either of the ZIP or CDF families—have not previously been identified plant vacuolar membranes.
PtdMTP1 mRNA was expressed constitutively throughout the hybrid poplar used in this study and was not expressed differentially by varying the Zn concentrations. Similarly, RNA gel blot experiments in Arabidopsis indicated that ZAT mRNA was expressed throughout the plant at uniform but low levels and was not induced by increasing the Zn concentrations (van der Zaal et al., 1999
To date, no clear picture has emerged regarding whether plant CDF transporters might energize metal transport through coupling to antiport with H+. Bloss et al. (2002)
Previous studies involving metal blots have suggested that AtZAT might form dimers (Bloss et al., 2002
Interestingly, we discovered a LZ motif in PtdMTP1. The LZ domain is a helical structure that forms coiled coils and was identified originally as a highly conserved motif mediating the binding of transcription factors to DNA (Landschulz et al., 1988
It appears that the fourth and last Leu residue (Leu-314) of the LZ motif is particularly critical for the function of PtdMTP1. Interestingly, this final Leu residue of the heptad repeat was found to be highly conserved among plant CDFs (Figure 10). Studies of the AtZAT protein have revealed that the C-terminal putative intracellular region is critical for activity, because truncation just after the last transmembrane domain was not able to confer Zn resistance or Zn accumulation in Arabidopsis (van der Zaal et al., 1999
PtdMTP1 possesses the CDF family–specific signature sequence (Paulsen and Saier, 1997
To breed plants with superior phytoremediation potential, one possible strategy is to enhance the biomass productivity of species that are hyperaccumulators. An alternative strategy is to enhance metal accumulation in high-biomass species (Pilon-Smits and Pilon, 2002
Plant Growth Conditions Hybrid poplar (Populus trichocarpa x Populus deltoides cv Beaupré) was grown in a greenhouse in a controlled environment as described previously (Kohler et al., 2003 ). Arabidopsis thaliana plants (ecotype Columbia) were grown in Klasman Substrate No. 1 compost (Klasmann-Deilmann, Geest, Germany) in a greenhouse at 23°C with a 16-h photoperiod. Alternatively, seeds were sterilized and grown at 22°C with a 16-h photoperiod on agar plates containing sterile MS medium (Murashige and Skoog, 1962). For Zn resistance assays of Arabidopsis lines expressing PtdMTP1 and lines transformed with the empty vector pART27, homozygous T3 seeds were sown on plates containing half-strength MS medium supplemented with various concentrations of ZnSO4. Wild-type plants were also used as a control. After 3 weeks of growth, plant fresh weights were determined.
Cloning of PtdMTP1, cDNA Array, and Reverse Transcriptase–PCR Analysis
Total RNA isolation was performed with the RNeasy Plant Mini kit (Qiagen, Courtab
Yeast Strains, Media, and Transformation
Site-Directed Mutagenesis
Construction of GFP and V5 Fusions for Expression in Yeast To construct PtdMTP1:GFP, the GFP (S65T) cDNA was amplified from the plasmid PFA6a-GFP65T-kanMX6 with the primers GFPf (5'-CCCCTCTAGAATTAACAGTAAAGGAGAAGAACTTTTCA-3') and GFPr (5'-CCCCTCTAGACTATTTGTATAGTTCATCCATGCC-3'), introducing a XbaI site at both ends (sites underlined), and cloned at the XbaI site in the pYES2 plasmid to generate a new plasmid called pYES2-GFP. Then, a PtdMTP1 fragment, lacking the stop codon of the PtdMTP1 cDNA, was amplified with the primers MTP1f and MTP1rnostop (5'-CCCCGGATCCCCACGCTCTATCTGGATGGTTACAT-3') (BamHI site underlined) and cloned into the HindIII-BamHI–digested pYES2-GFP plasmid. PtdMTP1:V5 was constructed by inserting the HindIII-BamHI fragment of PtdMTP1 into pYES6/CT (Invitrogen, Carlsbad, CA). Mutant PtdMTP1 clones also were fused in frame with GFP or V5 into the pYES2-GFP or pYES6/CT plasmid, respectively. As a control, the expression of nontargeted GFP in yeast was achieved by transforming cells with pYES2 in which only the full-length GFP cDNA was cloned.
Transient Expression of PtdMTP1 in Onion Cells
Generation of Transgenic Arabidopsis Expressing PtdMTP1 or PtdMTP1:GFP
Cell Labeling and Confocal Microscopy The GFP fluorescence of yeast and plant cells was visualized by confocal laser scanning microscopy (MRC-1000 [Bio-Rad] or Meta 510 [Zeiss, Jena, Germany]). Control cells did not show any green or red fluorescence for the settings at which images usually were collected. Digital images were recorded using the confocal microscope operating system software. For final processing, digital images were pseudocolored using Adobe Photoshop 4.0 (Mountain View, CA), with red attributed to FM4-64 or propidium iodide and green attributed to GFP, to correspond to real red and green colors. An image with transmitted light also was collected when needed using the confocal microscope.
Protein Extraction To obtain proteins of microsome fractions from plants expressing PtdMTP1:GFP, 3-week-old shoot tissues were ground in the presence of liquid nitrogen and homogenized subsequently with Tris buffer, pH 7.5, that contained 5 mM EGTA, 0.25% (v/v) Tween 20, 10 mM phenylmethylsulfonyl fluoride, and a cocktail of protease inhibitors (EDTA-free protease inhibitor cocktail tablets; Roche Diagnostics, Mannheim, Germany). The extracts were centrifuged first at 3,600g for 5 min to remove debris and subsequently at 13,000g for 15 min to pellet the microsome fraction. Finally, the proteins were homogenized in the extraction buffer. Even if an equivalent number of cells or mass of plant tissue was processed in each extraction, protein quantitation was performed with the Bio-Rad protein assay to standardize the extracts for protein concentration.
Protein Gel Blot Analysis Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Michel Chalot, michel.chalot{at}scbiol.uhp-nancy.fr.
Accession Numbers
We thank Legong Li and Sheng Luan (University of California, Berkeley) for providing Arabidopsis transgenic plants expressing AtMGT1 fused to GFP and Catherine Jauniaux (Institut de Biologie et de Médecine Moléculaires, Gosselies, Belgium) for providing the PFA6a-GFP65T-kanMX6 plasmid. We acknowledge Chris Winefield (University of York, UK) for the gift of pART7 and pART27 plasmids and for helpful discussions. We also thank Peter O'Toole (University of York) for providing help with confocal microscopy, Sylvain Jeandroz (University H. Poincaré, Nancy, France) for helping with phylogenetic analysis, Valérie Legué (University H. Poincaré) for providing access to microscopy and image-analysis facilities, Pascal Frey (Institut National de la Recherche Agronomique [INRA], Nancy, France) for the gift of poplar trees, and Jared Cartwright, Viktor Filatov, Frans Maathuis (University of York), and Jean Pierre Jacquot (University H. Poincaré) for stimulating discussions. The contributions of Jeremy Couturier and Régis Belleville are gratefully acknowledged. A.K. was supported by postdoctoral fellowships from the INRA and the Région de Lorraine. The present investigation was supported by grants from the INRA (Programmes Functional Genomics of Poplar and LIGNOME) and from the European Union, Framework Programme 5 (Metallophytes project).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017541.
1 Current address: Unité Mixte de Recherche Institut National de la Recherche Agronomique/Université Henri Poincaré 1136 Interactions Arbres/Micro-organismes, Faculté des Sciences, UHP Nancy I, BP 239, 54506 Vandoeuvre, France. Received September 18, 2003; accepted September 18, 2003.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410.[CrossRef][Web of Science][Medline] Bachhawat, A.K., Manolson, M.F., Murdock, D.G., Garman, J.D., and Jones, E.W. (1993). The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase. Yeast 9, 175–184.[CrossRef][Medline] Bloss, T., Clemens, S., and Nies, D.H. (2002). Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes. Planta 214, 783–791.[CrossRef][Web of Science][Medline]
Chardonnens, A.N., Koevoets, P.L., van Zanten, A., Schat, H., and Verkleij, J.A. (1999). Properties of enhanced tonoplast zinc transport in naturally selected zinc-tolerant Silene vulgaris. Plant Physiol. 120, 779–786. Clemens, S. (2001). Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212, 475–486.[CrossRef][Web of Science][Medline]
Clemens, S., Bloss, T., Vess, C., Neumann, D., Nies, D.H., and Zur Nieden, U. (2002a). A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance. J. Biol. Chem. 277, 18215–18221. Clemens, S., Palmgren, M.G., and Krämer, U. (2002b). A long way ahead: Understanding and engineering plant metal accumulation. Trends Plant Sci. 7, 309–315.[CrossRef][Web of Science][Medline] Clough, S.J., and Bent, A. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.[CrossRef][Web of Science][Medline]
Conklin, D.S., McMaster, J.A., Culbertson, M.R., and Kung, C. (1992). COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 3678–3688.
Delhaize, E., Kataoka, T., Hebb, D.M., White, R.G., and Ryan, P.R. (2003). Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance. Plant Cell 15, 1131–1142. Di Baccio, D., Tognetti, R., Sebastiani, L., and Vitagliano, C. (2003). Responses of Populus deltoides x Populus nigra (Populus x euramericana) clone I-214 to high zinc concentrations. New Phytol. 159, 443–452.[CrossRef] Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300, 1005–1016.[CrossRef][Web of Science][Medline] Gaither, L.A., and Eide, D.J. (2001). Zinc transporters and their regulation. Biometals 14, 251–270.[CrossRef][Web of Science][Medline]
Gietz, D., St. Jean, A., Woods, R.A., and Schiestl, R.H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425. Gleave, A.P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.[CrossRef][Web of Science][Medline] Gordon, M., Choe, N., Duffy, J., Ekuan, G., Heilman, P., Muiznieks, I., Ruszaj, M., Shurtleff, B.B., Strand, S., Wilmoth, J., and Newman, L.A. (1998). Phytoremediation of trichloroethylene with hybrid poplars. Environ. Health Perspect. 106, 1001–1004.
Graham, L.A., Hill, K.J., and Stevens, T.H. (1995). VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H+-ATPase required for function and assembly of the enzyme complex. J. Biol. Chem. 270, 15037–15044.
Haseloff, J., Siemering, K.R., Prasher, D.C., and Hodge, S. (1997). Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94, 2122–2127.
Huang, L., Kirschke, C.P., and Gitschier, J. (2002). Functional characterization of a novel mammalian zinc transporter, ZNT6. J. Biol. Chem. 277, 26389–26395.
Humair, D., Hernandez Felipe, D., Neuhaus, J.M., and Paris, N. (2001). Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor. Plant Cell 13, 781–792. Kamizono, A., Nishizawa, M., Teranishi, Y., Murata, K., and Kimura, A. (1989). Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 219, 161–167.[CrossRef][Web of Science][Medline]
Kirschke, C.P., and Huang, L. (2003). ZNT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. 278, 4096–4102. Kohler, A., Delaruelle, C., Martin, D., Encelot, N., and Martin, F. (2003). The poplar root transcriptome: Analysis of 7000 expressed sequence tags. FEBS Lett. 542, 37–41.[CrossRef][Web of Science][Medline]
Küpper, H., Zhao, F.J., and McGrath, S.P. (1999). Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiol. 119, 305–312. Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.[CrossRef][Web of Science][Medline]
Lacourt, I., Duplessis, S., Abba, S., Bonfante, P., and Martin, F. (2002). Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii. Appl. Environ. Microbiol. 68, 4574–4582. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Landschulz, W.H., Johnson, P.F., and McKnight, S.L. (1988). The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759–1764.
Lasat, M.M. (2002). Phytoextraction of toxic metals: A review of biological mechanisms. J. Environ. Qual. 31, 109–120.
Leung, I.W., and Lassam, N. (1998). Dimerization via tandem leucine zippers is essential for the activation of the mitogen-activated protein kinase kinase kinase, MLK-3. J. Biol. Chem. 273, 32408–32415.
Li, L., and Kaplan, J. (1998). Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity. J. Biol. Chem. 273, 22181–22187.
Li, L., and Kaplan, J. (2001). The yeast gene MSC2, a member of the cation diffusion facilitator family, affects the cellular distribution of zinc. J. Biol. Chem. 276, 5036–5043.
Li, L., Tutone, A.F., Drummond, R.S., Gardner, R.C., and Luan, S. (2001). A novel family of magnesium transport genes in Arabidopsis. Plant Cell 13, 2761–2775.
Li, Z.S., Lu, Y.P., Zhen, R.G., Szczypka, M., Thiele, D.J., and Rea, P.A. (1997). A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium. Proc. Natl. Acad. Sci. USA 94, 42–47.
Luo, Z., Matthews, A.M., and Weiss, S.R. (1999). Amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-to-cell fusion. J. Virol. 73, 8152–8159.
Lyons, T.J., Gasch, A.P., Gaither, L.A., Botstein, D., Brown, P.O., and Eide, D.J. (2000). Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast. Proc. Natl. Acad. Sci. USA 97, 7957–7962. MacDiarmid, C.W., Gaither, L.A., and Eide, D. (2000). Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae. EMBO J. 19, 2845–2855.[CrossRef][Web of Science][Medline]
MacDiarmid, C.W., Milanick, M.A., and Eide, D.J. (2002). Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J. Biol. Chem. 277, 39187–39194.
Marx, S.O., Reiken, S., Hisamatsu, Y., Gaburjakova, M., Gaburjakova, J., Yang, Y.M., Rosemblit, N., and Marks, A.R. (2001). Phosphorylation-dependent regulation of ryanodine receptors: A novel role for leucine/isoleucine zippers. J. Cell Biol. 153, 699–708.
Mäser, P., et al. (2001). Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol. 126, 1646–1667.
McCormack, K., Tanouye, M.A., Iverson, L.E., Lin, J.W., Ramaswami, M., McCormack, T., Campanelli, J.T., Mathew, M.K., and Rudy, B. (1991). A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels. Proc. Natl. Acad. Sci. USA 88, 2931–2935. Miyabe, S., Izawa, S., and Inoue, Y. (2001). The Zrc1 is involved in zinc transport system between vacuole and cytosol in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 282, 79–83.[CrossRef][Medline] Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497.[CrossRef] Palmiter, R.D., Cole, T.B., and Findley, S.D. (1996a). ZNT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J. 15, 1784–1791.[Web of Science][Medline]
Palmiter, R.D., Cole, T.B., Quaife, C.J., and Findley, S.D. (1996b). ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc. Natl. Acad. Sci. USA 93, 14934–14939. Palmiter, R.D., and Findley, S.D. (1995). Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 14, 639–649.[Web of Science][Medline] Paulsen, I.T., and Saier, M.H., Jr. (1997). A novel family of ubiquitous heavy metal ion transport proteins. J. Membr. Biol. 156, 99–103.[CrossRef][Web of Science][Medline]
Pence, N.S., Larsen, P.B., Ebbs, S.D., Letham, D.L., Lasat, M.M., Garvin, D.F., Eide, D., and Kochian, L.V. (2000). The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc. Natl. Acad. Sci. USA 97, 4956–4960.
Persans, M.W., Nieman, K., and Salt, D.E. (2001). Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense. Proc. Natl. Acad. Sci. USA 98, 9995–10000. Pilon-Smits, E., and Pilon, M. (2002). Phytoremediation of metals using transgenic plants. Crit. Rev. Plant Sci. 21, 439–456.[CrossRef] Ramsay, L.M., and Gadd, G.M. (1997). Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification. FEMS Microbiol. Lett. 152, 293–298.[CrossRef][Medline]
Reitter, J.N., Sergel, T., and Morrison, T.G. (1995). Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein. J. Virol. 69, 5995–6004. Rugh, C.L., Senecoff, J.F., Meagher, R.B., and Merkle, S.A. (1998). Development of transgenic yellow poplar for mercury phytoremediation. Nat. Biotechnol. 16, 925–928.[CrossRef][Web of Science][Medline] Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Scott, A., Wyatt, S., Tsou, P.L., Robertson, D., and Allen, N.S. (1999). System for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26, 1128–1132. Sherman, F. (1991). Getting started with yeast. Methods Enzymol. 28, 3–20.
Simmerman, H.K., Kobayashi, Y.M., Autry, J.M., and Jones, L.R. (1996). A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J. Biol. Chem. 271, 5941–5946. Stanton, B., Eaton, J., Johnson, J., Rice, D., Schuette, B., and Moser, B. (2002). Hybrid poplar in the Pacific Northwest: The effects of market-driven management. J. For. 100, 28–33.
Sterky, F., et al. (1998). Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags. Proc. Natl. Acad. Sci. USA 95, 13330–13335.
Supek, F., Supekova, L., Nelson, H., and Nelson, N. (1996). A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria. Proc. Natl. Acad. Sci. USA 93, 5105–5110.
Surks, H.K., Mochizuki, N., Kasai, Y., Georgescu, S.P., Tang, K.M., Ito, M., Lincoln, T.M., and Mendelsohn, M.E. (1999). Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase I alpha. Science 286, 1583–1587.
Szczypka, M.S., Wemmie, J.A., Moye-Rowley, W.S., and Thiele, D.J. (1994). A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein. J. Biol. Chem. 269, 22853–22857.
Taylor, G. (2002). Populus: Arabidopsis for forestry. Do we need a model tree? Ann. Bot. 90, 681–689.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. (1997). The CLUSTAL_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
Ton, V.K., Mandal, D., Vahadji, C., and Rao, R. (2002). Functional expression in yeast of the human secretory pathway Ca2+,Mn2+-ATPase defective in Hailey-Hailey disease. J. Biol. Chem. 277, 6422–6427.
Turner, R., and Tjian, R. (1989). Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science 243, 1689–1694.
van der Zaal, B.J., Neuteboom, L.W., Pinas, J.E., Chardonnens, A.N., Schat, H., Verkleij, J.A., and Hooykaas, P.J. (1999). Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation. Plant Physiol. 119, 1047–1055.
Vida, T.A., and Emr, S.D. (1995). New vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792. Williams, L.E., Pittman, J.K., and Hall, J.L. (2000). Emerging mechanisms for heavy metal transport in plants. Biochim. Biophys. Acta 1465, 104–126.[Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | THE PLANT CELL | PLANT PHYSIOLOGY | |
|---|---|---|---|
TOP
ABSTRACT
INTRODUCTION
uf, France) from 
