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Metal resistance in yeast mediated by the expression of a maize 20S proteasome α subunit
Gene 293 (2002) 199–204 www.elsevier.com/locate/gene
Metal resistance in yeast mediated by the expression of a maize 20S proteasome a subunit q Ce´line Forzani, Ste´phane Lobre´aux, Ste´phane Mari, Jean-Franc¸ois Briat, Michel Lebrun* Laboratoire de Biochimie et Physiologie Mole´culaire des Plantes, CNRS UMR 5004 Universite´ Montpellier 2, Agro-M/INRA, 2 place Viala, F-34060 Montpellier Cedex 1, France Received 5 February 2001; received in revised form 12 April 2002; accepted 3 June 2002 Received by W. Martin
Abstract Transformation of yeast cells with a maize cDNA ZmPAA, encoding a 20S proteasome a-subunit, conferred resistance to nickel, cadmium and cobalt. This resistance is not linked to a modification of the intracellular nickel content, as no accumulation of nickel was measured between yeast cells transformed with a void vector or the ZmPAA cDNA. The abundance of the ZmPAA mRNA was increased in the shoots of maize plants upon nickel treatment. These results suggest that the proteasome might be involved in nickel resistance by scavenging metal oxidized proteins both in plants and yeast. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Nickel; Stress; Protein degradation; Saccharomyces cerevisiae
1. Introduction Nickel is classified as an essential element for plant growth (Eskew et al., 1983). However, at high concentrations, nickel is an important environmental pollutant, which inhibits root elongation and affects the photosynthetic machinery leading to plant chlorosis (Marschner, 1995). Furthermore, nickel compounds are known human carcinogens (Costa, 1991). The mechanisms of nickel toxicity have not been well characterized. Nickel ions bind to lipids and proteins such as specific sub-sequences of histones (Bal et al., 1999, 2000) and as a result, induce oxidative damages leading to lipid peroxidation or crosslinking of proteins to DNA (Costa, 1991). Although nickel is not a direct catalyst of the Haber–Weiss reaction, it can bind to peptides containing histidine residues thus allowing hydrogen peroxide to oxidize nickel and promote the generation of oxygen free radicals (Toreilles et al., 1990). A major consequence of oxygen free radical damage to proteins is to target them for degradation by proteolysis (Davies et al., 1987). The degradation of abnormal proteins resulting from cadmium Abbreviations: SD medium, synthetic medium with dextrose; MM, minimum medium; bp, base pair(s) q ZmPAA cDNA GenBank accession number: AJ293343. * Corresponding author. Universite´ Montpellier 2, UMR 5004-CC002, 34095 Montpellier Cedex 5, France. Tel.: 133-4-6714-4799; fax: 133-46714-3637. E-mail address: [email protected] (M. Lebrun).
treatment in yeast is mediated in part by proteasome and by up-regulation of genes coding for the limiting components of the ubiquitin pathway (Jungmann et al., 1993). Moreover, the POH1 gene, coding a novel human 26S proteasome subunit, when over-expressed in cos-1 cells is able to confer resistance to various cytotoxic agents and ultra-violet light (Spataro et al., 1997). UV-damaged DNA binding proteins are targets of CUL4A-mediated ubiquitination and degradation. CUL4A is a member of the mammalian cullins family which are components of the E3 ubiquitin–ligase complex (Chen et al., 2001). These observations gave indications on the potential link between protection against various environmental injury, in particular those related to metal-mediated oxidative damages, and the proteasome, a multicatalytic protease complex involved in the degradation of abnormal proteins. Most of the time, proteins are first covalently attached to ubiquitin via the sequential action of an ubiquitin activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and, in some cases, an ubiquitin-protein ligase (E3) (Hershko and Ciechanover, 1998). The polyubiquitinated proteins are then degraded by the 26S proteasome complex in an ATP-dependent process (Voges et al., 1999). The 26S proteasome is composed of two sub-complexes, the 20S proteasome which contains the catalytic core of the protease and the 19/22S regulatory complexes. The 20S proteasome contains 14 different subunits which were classified in two families,
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00758-8
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a-type and b-type subunits, on the basis of their sequence similarities. The subunits are arranged in four rings, stacked together to form a hollow cylinder. The two outer rings contain seven different a subunits and the two inner rings seven different b subunits which are responsible for the proteolytic activities. This overall structure is conserved amongst all the eukaryotic cells analysed so far. In higher eukaryotic cells, proteasome is localized both in the cytoplasm and in the nucleus (Rubin and Finley, 1995). As stated above, very little is known about the molecular mechanisms involved in nickel resistance. A functional approach using Saccharomyces cerevisiae was pursued to uncover plant genes that confer nickel resistance when expressed in yeast. We report the isolation of a maize cDNA encoding an a-type subunit of the 20S proteasome which conferred nickel resistance when expressed in S. cerevisiae. 2. Materials and methods 2.1. Yeast strains and growth conditions The F113 (Mat a, can1, inol-13, ura3-52) yeast strain was grown at 30 8C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or in synthetic medium with dextrose (SD) (0.17% yeast nitrogen base without amino acid and ammonium sulfate (Difco), 2% dextrose, 0.5% ammonium sulfate) supplemented with the required amino acids. To assay metal resistance, cultures were grown to saturation for 24 h, in liquid SD medium. Cells were either diluted in SD liquid culture to an OD600: 0.01 or serial dilutions were spotted onto agar minimum medium with some modifications 0.5% ammonium sulfate as nitrogen source and BME vitamins from Gibco BRL, both media containing 0.5 mM NiSO4 or different metals at concentrations that inhibit yeast cell growth. 2.2. cDNA isolation and characterization The F113 yeast strain was transformed by the lithium acetate method with a maize root cDNA library (Loulergue et al., 1998). One million transformants isolated on SDuracil medium were pooled and stored at 280 8C in 15% glycerol. Two hundred fifty thousand transformants were then screened for nickel resistance. Plasmids were amplified in Escherichia coli strain JM109 [endA1, recA1, gyrA96, thi, hsdR17 (rk 2 , mk 1 ), relA1, supE44, D(lac-pro AB), F 0 , traD36, proAB, lacl qZDM15] (Sambrook et al., 1989). The cDNA inserts were sequenced by the dideoxynucleotide chain termination method. Sequence computer analysis was performed with DNA Strider software and by connecting to BLAST network service at NCBI (http://www.ncbi.nlm.nih.gov). Protein sequence alignments were generated by ClustalW (http://www2. ebi.ac.uk/clustalw/) and boxshade (http://www.ch.embnet. org/software/BOX - form.html) software.
2.3. Nickel content of yeast Yeast cells were grown for 24 h in SD medium containing various NiSO4 concentrations. Yeast cells were harvested by centrifugation and washed with 50 mM EDTA and then H2O. Cell pellets were mineralized in 500 ml of 6 M nitric acid at 100 8C for 2 h. After mineralization, the samples were diluted 10 times with water and flamed in a SpectrAA220 atomic absorption spectrometer. The nickel concentration is expressed in nmol per ml of yeast cell volume. Yeast cell volume was determined using an electronic particle Coulter Counter Chanelyser (ZM; Coultronics). 2.4. Maize plant cultures Maize plants (Zea mays L., var. AMO 406, Maı¨sadour, Mont-de-Marsan, France) were grown hydroponically as previously described (Thoiron and Briat, 1999). After 12 days of growth, 0.5 mM NiSO4 was added to the media. Roots and shoots were harvested separately after 5, 10, 24 and 48 h of nickel treatment and frozen at 280 8C until RNA extraction. 2.5. Northern blot analysis Total RNA was extracted using the guanidine method and Northern blotting was performed as described previously (Thoiron and Briat, 1999). Ten micrograms of total RNA were loaded per lane. Blots were hybridized with the fulllength ZmPAA cDNA probe and a 25S rRNA probe to assess RNA loading. Signals were detected by phosphor-Imager screen (Storm 860) and quantified with ImageQuant (Molecular Dynamics, Sunnyvale, CA). 3. Results 3.1. Isolation of a maize cDNA encoding a 20S proteasome a subunit We conducted a screen to isolate plant genes that confer nickel resistance to yeast cells. Saccharomyces cerevisiae was transformed with a maize root cDNA library constructed in the high copy number expression plasmid pYPGE15 (Loulergue et al., 1998). Yeast transformants were then selected for their ability to grow on a minimum medium containing 0.5 mM NiSO4. This is the minimum nickel concentration that totally inhibits yeast growth. Two hundred fifty thousand transformants were then screened for nickel resistance. After 5 days of growth at 30 8C, the colonies able to grow on this toxic medium were selected and further analysed. Plasmids were rescued from 98 independent resistant yeast transformants. After retransformation only seven cDNAs still conferred nickel tolerance to yeast. These cDNA inserts were sequenced and DNA databases were searched by the BLAST program.
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Fig. 1. Amino acid sequence alignments of the deduced ZmPAA protein (accession number: AJ293343) sequence with the other a proteasome subunits from rice (OsPAA1; AB026558), tobacco (NtPSA1;Y16644), Arabidopsis thaliana (AtPAA2; AF043519), human (HsPRCI; P34062) and yeast (ScPRCI; P21243). Identical amino acid residues appear in black boxes and similar ones in light boxes. The double line indicates residues characteristic. A putative NLS sequence is indicated by a single line and (X) marks conserved glycine residues.
One of the isolated cDNA encoded a deduced 247-aminoacid polypeptide with a predicted molecular mass of 27,386 Da (Fig. 1). The deduced amino acid sequence was similar to a-type 20S proteasome subunits of different organisms. The highest identities were found with OsPAA1 (96.4%) from rice (Sassa et al., 2000), NtPSA1 (86.6%) from tobacco (Bahrami and Gray, 1999), and AtPAA2 (85%) from Arabidopsis thaliana (Fu et al., 1998), which are all plant a-type 20S proteasome subunits. The maize cDNA was therefore named ZmPAA according to the A. thaliana nomenclature (Fu et al., 1998). The maize protein also showed 59.1% identity with PRCI (p27k) from human (Bey et al., 1993) and 48.2% identity with PRCI (Yc7a) from yeast (Fujiwara et al., 1990), both a-type 20S proteasome subunits. Further analysis of the maize peptide sequence identified a highly conserved motif at the N-terminus necessary for the assembly of the a ring (Groll et al., 1997) as well as conserved glycine residues. A putative nuclear localization sequence KKVPDK was found at position 54 and it has been suggested elsewhere that it could be involved in the nuclear import of the proteasome (Nederlof et al., 1995). All these structural elements strongly suggest
that the isolated maize cDNA encodes a ZmPAA 20S proteasome subunit. 3.2. Characterization of the metal-resistant phenotype conferred by the ZmPAA cDNA expression in yeast Fig. 2A shows that yeast cells transformed with the ZmPAA cDNA could grow on a minimum medium containing 0.6 mM NiSO4 compared to cells expressing the empty plasmid (pYPGE15). We then tested the specificity of the nickel-resistant phenotype by growing the transformed yeast cells on medium supplemented with growth inhibiting concentration of various metals. Fig. 2A shows that yeast cells expressing the ZmPAA cDNA were also resistant to toxic concentrations of cobalt and cadmium but not to zinc or copper. Analysis of the growth rates of yeast in liquid medium containing various metal concentrations confirms a clear resistance towards nickel, cobalt and cadmium and no resistance to zinc and copper, at all the metal concentrations tested (Fig. 2B). In order to understand how the ZmPAA protein could confer nickel resistance to yeast, the nickel content of trans-
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Fig. 2. Expression of the ZmPAA cDNA in yeast confers metal resistance. The F113 yeast strain was transformed with the ZmPAA cDNA (ZmPAA) and the empty plasmid pYPGE15 (pYPGE15). Yeast cultures were grown overnight in SD medium. (A) Five-fold serial dilutions were plated onto minimum medium. The plates were incubated for 5 days at 30 8C or (B) cells were either diluted in SD liquid containing culture to an OD600: 0.01. OD600 was measured after 24 h of growth. Both media contain various metal concentrations that inhibit yeast growth.
formed yeast cells was measured. Yeast cells expressing the void plasmid, pYPGE15 or the ZmPAA cDNA were exposed to various nickel concentrations. As shown in Fig. 3, ZmPAA transformed cells led to no change in the intracellular nickel concentration compared to control cells (pYPGE15). This result indicates that resistance is not linked to an alteration in intracellular nickel accumulation.
after 48 h. These results indicate that the level of ZmPAA mRNA in the shoots is inducible upon nickel treatment.
3.3. Induction of ZmPAA mRNA accumulation in response to nickel in maize plants We investigated the abundance of the ZmPAA mRNA in response to nickel exposure of maize plants. Northern blots were carried out with total RNA extracted from shoots and roots of maize plants, treated or not with 0.5 mM NiSO4 (Fig. 4A). The ZmPAA mRNA abundance was quantified relative to the 25S rRNA abundance (Fig. 4B). The ZmPAA mRNA abundance in the roots was slightly higher in the nickel-treated plants compared to the untreated control. However, in the shoots, the ZmPAA expression was clearly increased upon nickel treatment after 24 h compared to the non-treated plants and remained higher
Fig. 3. Intracellular nickel concentrations of transformed yeast. Yeast cells expressing the empty pYPGE15 plasmid or the ZmPAA cDNA were grown for 24 h in SD medium containing various nickel concentrations. The intracellular nickel content of yeast cells was determined by atomic absorption spectrometer. Values are the mean of three independent experiments and the bars represent the standard deviations.
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Fig. 4. Northern analysis of ZmPAA transcripts accumulation in maize plants. (A) Total RNA were extracted from shoots and roots of 12-dayold maize plants grown in the absence of nickel (0 metal) or in the presence of 0.5 mM NiSO4. RNA blots were hybridized with the full-length ZmPAA cDNA probe and a 25 S rRNA probe to control RNA loading. (B) Relative abundance of the ZmPAA transcripts quantified relatively to the 25S rRNA abundance.
4. Discussion In search for plant genes conferring nickel resistance, we expressed a maize root cDNA library in S. cerevisiae and screened for growth of transformed cells on a medium containing 0.5 mM NiSO4. We isolated a maize cDNA, ZmPAA, which encodes an a subunit of the 20S proteasome, sufficient to provide nickel resistance when expressed in yeast. Such a subunits are structurally and functionally conserved between the yeast S. cerevisiae and the plant A. thaliana (Fu et al., 1999). Several of the Arabidopsis a subunits (PAC1 and PAE1) were able to replace their yeast orthologues in functional complementation experiments (Fu et al., 1998). Hence, it can be suggested that the ZmPAA protein can function in yeast as a component of the proteasome complex. It has been demonstrated in S. cerevisiae that cadmium resistance could be mediated by an ubiquitin-dependent proteolysis (Jungmann et al., 1993). The results showed that different yeast mutants affected in the activity of either an ubiquitin-conjugating enzyme (ubc7) or an essential proteasome b-subunit (pre1) rendered yeast cells sensitive to cadmium but not to zinc, copper or lead. Interestingly, our results indicate that overexpression of the ZmPAA cDNA in
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yeast conferred resistance to nickel, cobalt and cadmium but not to zinc or copper. Therefore, we hypothesized that nickel as well as cobalt could share the same mechanisms of toxicity as cadmium. The 20S proteasome has been described as a secondary antioxidative defense mechanisms (Grune and Davies, 1997) which degrades nonfunctional oxidized proteins such as ferritins (Rudeck et al., 2000) or histones (Ullrich et al., 1999). No changes in the nickel content of transformed nickel-resistant yeast cells has been observed (Fig. 3). This result is in agreement with a possible mechanism of resistance counteracting the oxidative damage of proteins produced by nickel, rather than by a modification of the intracellular nickel concentrations. However, the proteolytic activity of the 26S proteasome separated on a glycerol gradient (Enenkel et al., 1998) from the yeast cells expressing ZmPAA was unchanged in comparison to the one of cells expressing the empty plasmid (data not shown). It does not seem, therefore, that the yeast orthologue of ZmPAA could encode a limiting factor which would be bypassed by overexpressing ZmPAA, leading to an increase in the proteolytic activity of the yeast 26S proteasome. Alternatively, inhibition of the proteasome function can lead to an increase in the survival of mammalian cells subjected to high temperature up to 46 8C (Bush et al., 1997) and other injury, though the lack of proteasome activity under such stress conditions should lead to a cellular accumulation of damaged proteins otherwise targeted for degradation. However, it does not seem that the expression of the ZmPAA subunit could act in yeast as a ‘dominant-negative’ (Herskowitz, 1987) inhibitory subunit since, again, no decrease in the proteolytic activity of the 26S proteasome was measured in response to ZmPAA expression (data not shown). In the absence of nickel treatment, ZmPAA mRNA accumulation was higher in the roots than in the shoots. Upon nickel treatment, a clear induction was observed in the shoots and was absent in the roots (Fig. 4). However, the accumulation of ZmPAA transcript in the roots was already very high before nickel treatment and probably reached saturation. Jungmann et al. (1993) have also shown in yeast that the ubiquitin-dependent proteolysis system was cadmium inducible. Therefore, the degradation of oxidized proteins by the proteasome is likely to play an important role in the resistance towards metal toxicity both in yeast and plants. In conclusion, we have isolated a maize cDNA encoding an a subunit from the 20S proteasome which confers nickel resistance in yeast. The 20S proteasome is likely to be involved in the removal of damaged proteins generated by nickel. The specific role of this ZmPAA a subunit in the 20S proteasome activity remains to be determined. Because we did not get an indication that the overexpression of this subunit modified the overall proteolytic activity of the 26S activity, it could be hypothesized that the ZmPAA subunit may interact with substrates outside the 20S proteasome. Alternatively, it could modify the subcellular distribution of the proteasome (Enenkel et al., 1998).
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Acknowledgements We thank Dr. Sylvie Dequin for help with counting yeast cell volume using a Coulter Counter Channelyser. References Bahrami, A.R., Gray, J.E., 1999. Expression of a proteasome alpha-type subunit gene during tobacco development and senescence. Plant Mol. Biol. 39, 325–333. Bal, W., Karantza, V., Moudrianakis, E.N., Kasprzak, K.S., 1999. Interaction of Nickel(II) with histones: in vitro binding of nickel(II) to the core histone tetramer. Arch. Biochem. Biophys. 364, 161–166. Bal, W., Liang, R., Lukszo, J., Lee, S.H., Dizdaroglu, M., Kasprzak, K.S., 2000. Ni(II) specifically cleaves the C-terminal tail of the major variant of histone H2A and forms an oxidative damage-mediating complex with the cleaved-off octapeptide. Chem. Res. Toxicol. 13, 616–624. Bey, F., Silva Pereira, I., Coux, O., Viegas-Pequignot, E., Recillas Targa, F., Nothwang, H.G., Dutrillaux, B., Scherrer, K., 1993. The prosomal RNA-binding protein p27K is a member of the alpha-type human prosomal gene family. Mol. Gen. Genet. 237, 193–205. Bush, K.T., Goldberg, A.L., Nigam, S.K., 1997. Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. J. Biol. Chem. 272, 9086–9092. Chen, X., Zhang, Y., Douglas, L., Zhou, P., 2001. UV-damaged DNAbinding proteins are targets of CUL-4A-mediated ubiquitination and degradation. J. Biol. Chem. 276, 48175–48182. Costa, M., 1991. Molecular mechanisms of nickel carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 31, 321–337. Davies, K.J., Lin, S.W., Pacifici, R.E., 1987. Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein. J. Biol. Chem. 262, 9914–9920. Enenkel, C., Lehmann, A., Kloetzel, P.M., 1998. Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast. EMBO J. 17, 6144–6154. Eskew, A.W., Welch, R.M., Carey, E.E., 1983. Nickel: an essential micronutrient for legumes and possibly all higher plants. Science 222, 621– 623. Fu, H., Doelling, J.H., Arendt, C.S., Hochstrasser, M., Vierstra, R.D., 1998. Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana. Genetics 149, 677–692. Fu, H., Girod, P.A., Doelling, J.H., van Nocker, S., Hochstrasser, M., Finley, D., Vierstra, R.D., 1999. Structure and functional analysis of the 26S proteasome subunits from plants. Mol. Biol. Rep. 26, 137– 146. Fujiwara, T., Tanaka, K., Orino, E., Yoshimura, T., Kumatori, A., Tamura, T., Chung, C.H., Nakai, T., Yamaguchi, K., Shin, S., Kakizuka, A., Nakanishi, S., Ichihara, A., 1990. Proteasomes are essential for yeast
proliferation: cDNA cloning and gene disruption of two major subunits. J. Biol. Chem. 265, 16604–16613. Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, H.D., ˚ Huber, R., 1997. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463–471. Grune, T., Davies, K.J., 1997. Breakdown of oxidized proteins as a part of secondary antioxidant defenses in mammalian cells. Biofactors 6, 165– 172. Hershko, A., Ciechanover, A., 1998. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479. Herskowitz, I., 1987. Functional inactivation of genes by dominant negative mutations. Nature 329, 219–222. Jungmann, J., Reins, H.A., Schobert, C., Jentsch, S., 1993. Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature 361, 369–371. Loulergue, C., Lebrun, M., Briat, J.F., 1998. Expression cloning in Fe 21 transport defective yeast of a novel maize MYC transcription factor. Gene 225, 47–57. Marschner, H., 1995. Mineral Nutrition of Higher Plants, 2nd Edition. Academic Press, London. Nederlof, P.M., Wang, H.R., Baumeister, W., 1995. Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro. Proc. Natl. Acad. Sci. USA 92, 12060–12064. Rubin, D.M., Finley, D., 1995. Proteolysis. The proteasome: a proteindegrading organelle? Curr. Biol. 5, 854–858. Rudeck, M., Volk, T., Sitte, N., Grune, T., 2000. Ferritin oxidation in vitro: implication of iron release and degradation by the 20S proteasome. IUBMB Life 49, 451–456. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sassa, H., Oguchi, S., Inoue, T., Hirano, H., 2000. Primary structural features of the 20S proteasome subunits of rice (Oryza sativa). Gene 250, 61–66. Spataro, V., Toda, T., Craig, R., Seeger, M., Dubiel, W., Harris, A.L., Norbury, C., 1997. Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit. J. Biol. Chem. 272, 30470–30475. Thoiron, S., Briat, J.-F., 1999. Differential expression of maize sugar responsive genes in response to iron deficiency. Plant Physiol. Biochem. 37, 759–766. Toreilles, J., Gue´ rin, M.-C., Slaoui-Hasnaoui, A., 1990. Nickel (II) complexes of histidyl-peptides as fenton-reaction catalysts. Free Radic. Res. Commun. 11, 159–166. Ullrich, O., Sitte, N., Sommerburg, O., Sandig, V., Davies, K.J., Grune, T., 1999. Influence of DNA binding on the degradation of oxidized histones by the 20S proteasome. Arch. Biochem. Biophys. 362, 211–216. Voges, D., Zwickl, P., Baumeister, W., 1999. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068.
Metal resistance in yeast mediated by the expression of a maize 20S proteasome a subunit q Ce´line Forzani, Ste´phane Lobre´aux, Ste´phane Mari, Jean-Franc¸ois Briat, Michel Lebrun* Laboratoire de Biochimie et Physiologie Mole´culaire des Plantes, CNRS UMR 5004 Universite´ Montpellier 2, Agro-M/INRA, 2 place Viala, F-34060 Montpellier Cedex 1, France Received 5 February 2001; received in revised form 12 April 2002; accepted 3 June 2002 Received by W. Martin
Abstract Transformation of yeast cells with a maize cDNA ZmPAA, encoding a 20S proteasome a-subunit, conferred resistance to nickel, cadmium and cobalt. This resistance is not linked to a modification of the intracellular nickel content, as no accumulation of nickel was measured between yeast cells transformed with a void vector or the ZmPAA cDNA. The abundance of the ZmPAA mRNA was increased in the shoots of maize plants upon nickel treatment. These results suggest that the proteasome might be involved in nickel resistance by scavenging metal oxidized proteins both in plants and yeast. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Nickel; Stress; Protein degradation; Saccharomyces cerevisiae
1. Introduction Nickel is classified as an essential element for plant growth (Eskew et al., 1983). However, at high concentrations, nickel is an important environmental pollutant, which inhibits root elongation and affects the photosynthetic machinery leading to plant chlorosis (Marschner, 1995). Furthermore, nickel compounds are known human carcinogens (Costa, 1991). The mechanisms of nickel toxicity have not been well characterized. Nickel ions bind to lipids and proteins such as specific sub-sequences of histones (Bal et al., 1999, 2000) and as a result, induce oxidative damages leading to lipid peroxidation or crosslinking of proteins to DNA (Costa, 1991). Although nickel is not a direct catalyst of the Haber–Weiss reaction, it can bind to peptides containing histidine residues thus allowing hydrogen peroxide to oxidize nickel and promote the generation of oxygen free radicals (Toreilles et al., 1990). A major consequence of oxygen free radical damage to proteins is to target them for degradation by proteolysis (Davies et al., 1987). The degradation of abnormal proteins resulting from cadmium Abbreviations: SD medium, synthetic medium with dextrose; MM, minimum medium; bp, base pair(s) q ZmPAA cDNA GenBank accession number: AJ293343. * Corresponding author. Universite´ Montpellier 2, UMR 5004-CC002, 34095 Montpellier Cedex 5, France. Tel.: 133-4-6714-4799; fax: 133-46714-3637. E-mail address: [email protected] (M. Lebrun).
treatment in yeast is mediated in part by proteasome and by up-regulation of genes coding for the limiting components of the ubiquitin pathway (Jungmann et al., 1993). Moreover, the POH1 gene, coding a novel human 26S proteasome subunit, when over-expressed in cos-1 cells is able to confer resistance to various cytotoxic agents and ultra-violet light (Spataro et al., 1997). UV-damaged DNA binding proteins are targets of CUL4A-mediated ubiquitination and degradation. CUL4A is a member of the mammalian cullins family which are components of the E3 ubiquitin–ligase complex (Chen et al., 2001). These observations gave indications on the potential link between protection against various environmental injury, in particular those related to metal-mediated oxidative damages, and the proteasome, a multicatalytic protease complex involved in the degradation of abnormal proteins. Most of the time, proteins are first covalently attached to ubiquitin via the sequential action of an ubiquitin activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and, in some cases, an ubiquitin-protein ligase (E3) (Hershko and Ciechanover, 1998). The polyubiquitinated proteins are then degraded by the 26S proteasome complex in an ATP-dependent process (Voges et al., 1999). The 26S proteasome is composed of two sub-complexes, the 20S proteasome which contains the catalytic core of the protease and the 19/22S regulatory complexes. The 20S proteasome contains 14 different subunits which were classified in two families,
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00758-8
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a-type and b-type subunits, on the basis of their sequence similarities. The subunits are arranged in four rings, stacked together to form a hollow cylinder. The two outer rings contain seven different a subunits and the two inner rings seven different b subunits which are responsible for the proteolytic activities. This overall structure is conserved amongst all the eukaryotic cells analysed so far. In higher eukaryotic cells, proteasome is localized both in the cytoplasm and in the nucleus (Rubin and Finley, 1995). As stated above, very little is known about the molecular mechanisms involved in nickel resistance. A functional approach using Saccharomyces cerevisiae was pursued to uncover plant genes that confer nickel resistance when expressed in yeast. We report the isolation of a maize cDNA encoding an a-type subunit of the 20S proteasome which conferred nickel resistance when expressed in S. cerevisiae. 2. Materials and methods 2.1. Yeast strains and growth conditions The F113 (Mat a, can1, inol-13, ura3-52) yeast strain was grown at 30 8C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or in synthetic medium with dextrose (SD) (0.17% yeast nitrogen base without amino acid and ammonium sulfate (Difco), 2% dextrose, 0.5% ammonium sulfate) supplemented with the required amino acids. To assay metal resistance, cultures were grown to saturation for 24 h, in liquid SD medium. Cells were either diluted in SD liquid culture to an OD600: 0.01 or serial dilutions were spotted onto agar minimum medium with some modifications 0.5% ammonium sulfate as nitrogen source and BME vitamins from Gibco BRL, both media containing 0.5 mM NiSO4 or different metals at concentrations that inhibit yeast cell growth. 2.2. cDNA isolation and characterization The F113 yeast strain was transformed by the lithium acetate method with a maize root cDNA library (Loulergue et al., 1998). One million transformants isolated on SDuracil medium were pooled and stored at 280 8C in 15% glycerol. Two hundred fifty thousand transformants were then screened for nickel resistance. Plasmids were amplified in Escherichia coli strain JM109 [endA1, recA1, gyrA96, thi, hsdR17 (rk 2 , mk 1 ), relA1, supE44, D(lac-pro AB), F 0 , traD36, proAB, lacl qZDM15] (Sambrook et al., 1989). The cDNA inserts were sequenced by the dideoxynucleotide chain termination method. Sequence computer analysis was performed with DNA Strider software and by connecting to BLAST network service at NCBI (http://www.ncbi.nlm.nih.gov). Protein sequence alignments were generated by ClustalW (http://www2. ebi.ac.uk/clustalw/) and boxshade (http://www.ch.embnet. org/software/BOX - form.html) software.
2.3. Nickel content of yeast Yeast cells were grown for 24 h in SD medium containing various NiSO4 concentrations. Yeast cells were harvested by centrifugation and washed with 50 mM EDTA and then H2O. Cell pellets were mineralized in 500 ml of 6 M nitric acid at 100 8C for 2 h. After mineralization, the samples were diluted 10 times with water and flamed in a SpectrAA220 atomic absorption spectrometer. The nickel concentration is expressed in nmol per ml of yeast cell volume. Yeast cell volume was determined using an electronic particle Coulter Counter Chanelyser (ZM; Coultronics). 2.4. Maize plant cultures Maize plants (Zea mays L., var. AMO 406, Maı¨sadour, Mont-de-Marsan, France) were grown hydroponically as previously described (Thoiron and Briat, 1999). After 12 days of growth, 0.5 mM NiSO4 was added to the media. Roots and shoots were harvested separately after 5, 10, 24 and 48 h of nickel treatment and frozen at 280 8C until RNA extraction. 2.5. Northern blot analysis Total RNA was extracted using the guanidine method and Northern blotting was performed as described previously (Thoiron and Briat, 1999). Ten micrograms of total RNA were loaded per lane. Blots were hybridized with the fulllength ZmPAA cDNA probe and a 25S rRNA probe to assess RNA loading. Signals were detected by phosphor-Imager screen (Storm 860) and quantified with ImageQuant (Molecular Dynamics, Sunnyvale, CA). 3. Results 3.1. Isolation of a maize cDNA encoding a 20S proteasome a subunit We conducted a screen to isolate plant genes that confer nickel resistance to yeast cells. Saccharomyces cerevisiae was transformed with a maize root cDNA library constructed in the high copy number expression plasmid pYPGE15 (Loulergue et al., 1998). Yeast transformants were then selected for their ability to grow on a minimum medium containing 0.5 mM NiSO4. This is the minimum nickel concentration that totally inhibits yeast growth. Two hundred fifty thousand transformants were then screened for nickel resistance. After 5 days of growth at 30 8C, the colonies able to grow on this toxic medium were selected and further analysed. Plasmids were rescued from 98 independent resistant yeast transformants. After retransformation only seven cDNAs still conferred nickel tolerance to yeast. These cDNA inserts were sequenced and DNA databases were searched by the BLAST program.
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Fig. 1. Amino acid sequence alignments of the deduced ZmPAA protein (accession number: AJ293343) sequence with the other a proteasome subunits from rice (OsPAA1; AB026558), tobacco (NtPSA1;Y16644), Arabidopsis thaliana (AtPAA2; AF043519), human (HsPRCI; P34062) and yeast (ScPRCI; P21243). Identical amino acid residues appear in black boxes and similar ones in light boxes. The double line indicates residues characteristic. A putative NLS sequence is indicated by a single line and (X) marks conserved glycine residues.
One of the isolated cDNA encoded a deduced 247-aminoacid polypeptide with a predicted molecular mass of 27,386 Da (Fig. 1). The deduced amino acid sequence was similar to a-type 20S proteasome subunits of different organisms. The highest identities were found with OsPAA1 (96.4%) from rice (Sassa et al., 2000), NtPSA1 (86.6%) from tobacco (Bahrami and Gray, 1999), and AtPAA2 (85%) from Arabidopsis thaliana (Fu et al., 1998), which are all plant a-type 20S proteasome subunits. The maize cDNA was therefore named ZmPAA according to the A. thaliana nomenclature (Fu et al., 1998). The maize protein also showed 59.1% identity with PRCI (p27k) from human (Bey et al., 1993) and 48.2% identity with PRCI (Yc7a) from yeast (Fujiwara et al., 1990), both a-type 20S proteasome subunits. Further analysis of the maize peptide sequence identified a highly conserved motif at the N-terminus necessary for the assembly of the a ring (Groll et al., 1997) as well as conserved glycine residues. A putative nuclear localization sequence KKVPDK was found at position 54 and it has been suggested elsewhere that it could be involved in the nuclear import of the proteasome (Nederlof et al., 1995). All these structural elements strongly suggest
that the isolated maize cDNA encodes a ZmPAA 20S proteasome subunit. 3.2. Characterization of the metal-resistant phenotype conferred by the ZmPAA cDNA expression in yeast Fig. 2A shows that yeast cells transformed with the ZmPAA cDNA could grow on a minimum medium containing 0.6 mM NiSO4 compared to cells expressing the empty plasmid (pYPGE15). We then tested the specificity of the nickel-resistant phenotype by growing the transformed yeast cells on medium supplemented with growth inhibiting concentration of various metals. Fig. 2A shows that yeast cells expressing the ZmPAA cDNA were also resistant to toxic concentrations of cobalt and cadmium but not to zinc or copper. Analysis of the growth rates of yeast in liquid medium containing various metal concentrations confirms a clear resistance towards nickel, cobalt and cadmium and no resistance to zinc and copper, at all the metal concentrations tested (Fig. 2B). In order to understand how the ZmPAA protein could confer nickel resistance to yeast, the nickel content of trans-
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Fig. 2. Expression of the ZmPAA cDNA in yeast confers metal resistance. The F113 yeast strain was transformed with the ZmPAA cDNA (ZmPAA) and the empty plasmid pYPGE15 (pYPGE15). Yeast cultures were grown overnight in SD medium. (A) Five-fold serial dilutions were plated onto minimum medium. The plates were incubated for 5 days at 30 8C or (B) cells were either diluted in SD liquid containing culture to an OD600: 0.01. OD600 was measured after 24 h of growth. Both media contain various metal concentrations that inhibit yeast growth.
formed yeast cells was measured. Yeast cells expressing the void plasmid, pYPGE15 or the ZmPAA cDNA were exposed to various nickel concentrations. As shown in Fig. 3, ZmPAA transformed cells led to no change in the intracellular nickel concentration compared to control cells (pYPGE15). This result indicates that resistance is not linked to an alteration in intracellular nickel accumulation.
after 48 h. These results indicate that the level of ZmPAA mRNA in the shoots is inducible upon nickel treatment.
3.3. Induction of ZmPAA mRNA accumulation in response to nickel in maize plants We investigated the abundance of the ZmPAA mRNA in response to nickel exposure of maize plants. Northern blots were carried out with total RNA extracted from shoots and roots of maize plants, treated or not with 0.5 mM NiSO4 (Fig. 4A). The ZmPAA mRNA abundance was quantified relative to the 25S rRNA abundance (Fig. 4B). The ZmPAA mRNA abundance in the roots was slightly higher in the nickel-treated plants compared to the untreated control. However, in the shoots, the ZmPAA expression was clearly increased upon nickel treatment after 24 h compared to the non-treated plants and remained higher
Fig. 3. Intracellular nickel concentrations of transformed yeast. Yeast cells expressing the empty pYPGE15 plasmid or the ZmPAA cDNA were grown for 24 h in SD medium containing various nickel concentrations. The intracellular nickel content of yeast cells was determined by atomic absorption spectrometer. Values are the mean of three independent experiments and the bars represent the standard deviations.
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Fig. 4. Northern analysis of ZmPAA transcripts accumulation in maize plants. (A) Total RNA were extracted from shoots and roots of 12-dayold maize plants grown in the absence of nickel (0 metal) or in the presence of 0.5 mM NiSO4. RNA blots were hybridized with the full-length ZmPAA cDNA probe and a 25 S rRNA probe to control RNA loading. (B) Relative abundance of the ZmPAA transcripts quantified relatively to the 25S rRNA abundance.
4. Discussion In search for plant genes conferring nickel resistance, we expressed a maize root cDNA library in S. cerevisiae and screened for growth of transformed cells on a medium containing 0.5 mM NiSO4. We isolated a maize cDNA, ZmPAA, which encodes an a subunit of the 20S proteasome, sufficient to provide nickel resistance when expressed in yeast. Such a subunits are structurally and functionally conserved between the yeast S. cerevisiae and the plant A. thaliana (Fu et al., 1999). Several of the Arabidopsis a subunits (PAC1 and PAE1) were able to replace their yeast orthologues in functional complementation experiments (Fu et al., 1998). Hence, it can be suggested that the ZmPAA protein can function in yeast as a component of the proteasome complex. It has been demonstrated in S. cerevisiae that cadmium resistance could be mediated by an ubiquitin-dependent proteolysis (Jungmann et al., 1993). The results showed that different yeast mutants affected in the activity of either an ubiquitin-conjugating enzyme (ubc7) or an essential proteasome b-subunit (pre1) rendered yeast cells sensitive to cadmium but not to zinc, copper or lead. Interestingly, our results indicate that overexpression of the ZmPAA cDNA in
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yeast conferred resistance to nickel, cobalt and cadmium but not to zinc or copper. Therefore, we hypothesized that nickel as well as cobalt could share the same mechanisms of toxicity as cadmium. The 20S proteasome has been described as a secondary antioxidative defense mechanisms (Grune and Davies, 1997) which degrades nonfunctional oxidized proteins such as ferritins (Rudeck et al., 2000) or histones (Ullrich et al., 1999). No changes in the nickel content of transformed nickel-resistant yeast cells has been observed (Fig. 3). This result is in agreement with a possible mechanism of resistance counteracting the oxidative damage of proteins produced by nickel, rather than by a modification of the intracellular nickel concentrations. However, the proteolytic activity of the 26S proteasome separated on a glycerol gradient (Enenkel et al., 1998) from the yeast cells expressing ZmPAA was unchanged in comparison to the one of cells expressing the empty plasmid (data not shown). It does not seem, therefore, that the yeast orthologue of ZmPAA could encode a limiting factor which would be bypassed by overexpressing ZmPAA, leading to an increase in the proteolytic activity of the yeast 26S proteasome. Alternatively, inhibition of the proteasome function can lead to an increase in the survival of mammalian cells subjected to high temperature up to 46 8C (Bush et al., 1997) and other injury, though the lack of proteasome activity under such stress conditions should lead to a cellular accumulation of damaged proteins otherwise targeted for degradation. However, it does not seem that the expression of the ZmPAA subunit could act in yeast as a ‘dominant-negative’ (Herskowitz, 1987) inhibitory subunit since, again, no decrease in the proteolytic activity of the 26S proteasome was measured in response to ZmPAA expression (data not shown). In the absence of nickel treatment, ZmPAA mRNA accumulation was higher in the roots than in the shoots. Upon nickel treatment, a clear induction was observed in the shoots and was absent in the roots (Fig. 4). However, the accumulation of ZmPAA transcript in the roots was already very high before nickel treatment and probably reached saturation. Jungmann et al. (1993) have also shown in yeast that the ubiquitin-dependent proteolysis system was cadmium inducible. Therefore, the degradation of oxidized proteins by the proteasome is likely to play an important role in the resistance towards metal toxicity both in yeast and plants. In conclusion, we have isolated a maize cDNA encoding an a subunit from the 20S proteasome which confers nickel resistance in yeast. The 20S proteasome is likely to be involved in the removal of damaged proteins generated by nickel. The specific role of this ZmPAA a subunit in the 20S proteasome activity remains to be determined. Because we did not get an indication that the overexpression of this subunit modified the overall proteolytic activity of the 26S activity, it could be hypothesized that the ZmPAA subunit may interact with substrates outside the 20S proteasome. Alternatively, it could modify the subcellular distribution of the proteasome (Enenkel et al., 1998).
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