Institute for Biochemistry, Ludwig Maximilians University of Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany
Correspondence
Andreas Uldschmid
uldschmi{at}biochem.mpg.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession number for the Trametes versicolor ctaA gene sequence reported in this article is AY210894.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to filamentous fungal systems, copper metabolism in yeast systems is well characterized: after being taken up from the medium by copper permeases, copper ions destined for insertion into secreted enzymes are bound by the copper chaperone Atx1p and transported to the post-Golgi compartment of the secretory pathway. There, Atx1p interacts with Ccc2p, a copper-transporting P-type ATPase, located in the membrane of the post-Golgi compartment. Ccc2p pumps the copper ions delivered by Atx1p into the lumen of the Golgi where copper is then inserted into secreted copper-dependent enzymes such as the iron oxidase Fet3p. The protein factors involved in cellular copper homeostasis are highly conserved between all eukaryotes analysed so far: copper metallochaperones homologous to Atx1p and to the copper-transporting P-type ATPases have been described in yeast, mammalian, nematode and plant systems (Himelblau et al., 1998; Hung et al., 1997
; Klomp et al., 1997
; Payne & Gitlin, 1998
; Hirayama et al., 1999
; Wakabayashi et al., 1998
). However, these factors seem to be regulated differently in the various systems.
Yeast mutants have facilitated the identification of genes from heterologous sources by functional complementation. Applying this to the secretory copper-containing laccases, we recently described the isolation, identification and characterization of a homologue of the yeast gene anti-oxidant 1 (ATX1), the gene tahA (Trametes ATX homologue) from Trametes versicolor encoding a protein of 72 aa with 56 % sequence identity to yeast Atx1p (Uldschmid et al., 2002). We observed that tahA was up-regulated when copper was present and down-regulated when no copper was available, and that this copper chaperone protein efficiently provided the secretory copper-containing laccases with copper.
Apart from providing a useful marker for monitoring copper trafficking to secretory pathways via the Golgi apparatus, the laccases are involved in a wide variety of different cellular reactions. Besides detoxification of toxic compounds, morphogenesis and melanin formation, laccases are regarded as potential pathogenic factors of fungi. They are also of practical interest because they can oxidize a wide variety of phenolic compounds, and can be used in paper-bleaching processes without chlorine (Call & Mücke, 1997), detoxification of xenobiotic compounds, organic synthesis (Rosenau et al., 1996
; Sariaslani, 1989
), dye bleaching, or binding of wood composites (Felby et al., 1997
). The production of functional laccases depends on sufficient/elevated copper concentrations in the medium (A. Uldschmid, R. Dombi & K. Marbach, unpublished data).
To further investigate copper trafficking to the laccases, we employed complementation strategies in yeast. Here we describe the identification of a second copper homeostasis factor, the gene ctaA (copper-transporting ATPase) from the basidiomycete T. versicolor encoding a protein of 983 aa with 40 % sequence identity to yeast Ccc2p. We characterized this gene and its product at the structural, biochemical and functional levels. We also studied the effect of ctaA overexpression on the amount of active laccase produced in yeast and Trametes, as well as the effect of coexpressing ctaA and the previously identified tahA gene. Interestingly, the effects of these gene products proved additive in both organisms, leading to elevated amounts of active laccase production, even under elevated copper conditions where, normally, copper systems are repressed and copper detoxification processes are switched on.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monokaryotic strains Trametes F6-79B11 (pyrG), used for cotransformation, and Trametes F2-100, used for Southern blot analyses, were derived from fruiting bodies of dikaryotic strains. DNA for library construction (cDNA and genomic) was obtained from dikaryotic TV-1 (Uldschmid et al., 2002
). Culture conditions for T. versicolor were as described by Uldschmid et al. (2002)
.
Plasmids and DNA libraries.
A genomic DNA library from T. versicolor was obtained from Dr Rupert Pfaller from the Consortium für elektrochemische Industrie, München. The library was constructed as described by Uldschmid et al. (2002). A Trametes cDNA library for yeast complementation was constructed in the yeast centromere plasmid pAH (Feldmann et al., 1996
), essentially as described by Uldschmid et al. (2002)
. Restriction analysis of inserts from different clones revealed that the cDNA library contains inserts ranging in size from 0·1 to 3·5 kb. For expression of Trametes genes in S. cerevisiae, the yeast expression vectors pAH, pAT (Feldmann et al., 1996
) and pYEX-S1 (Clontech) were used.
Isolation of T. versicolor ctaA cDNA by complementation.
Yeast Strain 3 was transformed with the pAH cDNA library (above) and, after plating out on His- selection medium, the 2x106 yeast clones obtained were washed off the plate, purified and thoroughly mixed before plating on selection plates containing 1 mM Ferrozine to create an iron deficiency and incubation for 3 days at 30 °C. From the 1000 colonies that grew on the selection plates, 70 were re-plated on His plates. The plasmids isolated from these clones were amplified in E. coli and the DNA analysed by restriction digests using HindIII.
Isolation of T. versicolor ctaA structural gene.
A complete genomic structural gene of ctaA-1 was obtained by PCR with the primers gcta-fw (5'-GAA TGC TGA AGT CGG GAG AAG C-3') and gcta-rv (5'-GAA GTT GAA CAT CCC GTG TGA C-3') and 500 ng of genomic DNA from T. versicolor with the Expand High Fidelity PCR system (Roche). The PCR conditions used were: 3 min at 94 °C, followed by 30 cycles of 10 s at 94 °C, 10 s at 60 °C and 4 min at 68 °C. The 4·9 kb product was separated on a 1 % agarose gel, extracted from the agarose using a Gel-Extraction Kit (Qiagen), ligated into the vector pCR2.1 and transformed into E. coli XL-1 Blue MRF'. Plasmid DNA was isolated from transformants and a correct clone (pCR2.1gcta) identified by restriction analysis.
Coexpression of tahA, ctaA and laccase in S. cerevisiae.
The laccase III gene (lacIII) from T. versicolor was coexpressed with ctaA, tahA or ctaA plus tahA in S. cerevisiae strain CM3260. Strain CM3260, which lacks endogenous laccase, was transformed with the laccase expression construct pLacP2 (lacIII cloned in the vector pYEX-S1 from Clontech; Fig. 2) where the lacIII cDNA was under transcriptional control of the yeast PGK promoter. This laccase-expressing yeast derivative was transformed with the pAH and pAT derivatives (Feldmann et al., 1996
) that harboured either the Trametes ctaA or tahA cDNA under control of the yeast ADH1 promoter (Figs 2 and 3
). The four strains were inoculated into 200 µl YNB selection medium (His-, Trp-, Ura-) containing 1 mM ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], supplemented with 0, 10, 100, 200, 400, 600, 800 or 1000 µM CuSO4, and grown in microtitre plates overnight, with shaking on a thermoshaker (Eppendorf) at room temperature. Laccase activity was then determined by measuring the OD420 value.
|
|
The gapDH promoter from pUC-Pgap- was excised with EcoRI and BspLU11I and inserted upstream of tahA via EcoRI and NcoI sites in the vector pZ-gTAH-N, resulting in the construct pZ-gTAH-Pgap. Subsequently, the gapDH promoter together with tahA was excised with EcoRI and NotI, the ends filled in, and this fragment cloned into the SmaI site of the plasmid gPura, carrying the Schizophyllum commune pyrG gene (Froelinger et al., 1989) under the control of the Trametes gapDH promoter (obtained from Dr Pfaller), resulting in the tahA expression vector gPuragapTAH (Fig. 5
).
|
Cotransformation of ctaA and tahA in T. versicolor.
Plasmids pUC-gapCTA and gPuragapTAH were mixed in a 1 : 1 ratio, transformed into T. versicolor F6-79B11 according to the protocol of Bartholomew et al. (2000) and plated out on minimal medium (MM) (Uldschmid et al., 2002
) containing 0·6 M sucrose. After 12 weeks incubation at 28 °C, the fungal colonies that appeared were inoculated on MM plates and kept at 28 °C until they were fully grown (
7 days). From the circumference region of the plates, mycelia were again inoculated on MM plates and incubated again until they had grown.
Some mycelium was scraped from each of the transformants and resuspended in 50 µl TE and prepared for a PCR with the following programme: 5 min at 65 °C2 min at 96 °C
4 min at 65 °C
1 min at 96 °C
1 min at 65 °C
0·5 min at 96 °C
20 °C. Subsequently, the cells were mechanically squashed with an inoculating loop and then centrifuged. Aliquots (1 µl) of supernatant were mixed with the primer pairs Pgap/tahE or Pgap/ctaA and Taq polymerase. The primer Pgap hybridizes in the gapDH promoter, tahE in the tahA gene and ctaA in the ctaA gene (see Fig. 5
). Thus the primer combinations were specific for the expression constructs. The PCR product with Pgap/tahE was 690 bp in size and that with Pgap/ctaA was 460 bp in size. As a positive control for the PCR, a 260 bp fragment was amplified out of the chromosomal T. versicolor gapDH gene with the primer pair Pgap/gapA.
Preparation of Trametes DNA and Southern blot analysis.
Preparation of genomic DNA from T. versicolor and analyses of yeast or T. versicolor transformants by Southern blotting were carried out as described by Uldschmid et al. (2002). DNA from cotransformants Ko7, Ko15 and Ko19 and the recipient strain F6 were cut with BamHI/NsiI, KpnI, SacI and XhoI, and the blot was hybridized with a tahA probe (NcoINsiI fragment from pZgTAH-N) or a ctaA probe (ClaINdeI fragment from pgATGcta). The BamHI/NsiI digest generated a 1·5 kb band containing the complete expression cassette with the gapDH promoter and tahA gene.
Isolation of mRNA from T. versicolor for library construction.
Total RNA from T. versicolor was prepared according to the method of Logemann et al. (1987), starting with 2 g semi-dried mycelium as described by Uldschmid et al. (2002)
.
Isolation of genes from a Trametes genomic library.
The Trametes genomic library was screened as described by Uldschmid et al. (2002).
Sequence analysis.
Clones were sequenced completely from their 5'- and 3'-ends by cycle sequencing on an automated sequencer (Laboratorium für molekulare Biologie, Abt. Genomics, München). Database searches with DNA fragment-derived protein sequence data were performed with the program BLASTX (National Institutes of Health). Alignments were generated using the CLUSTAL_X.63b Multiple Sequence Alignment Program, a multiple alignment tool developed by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Further sequence analysis and data processing were performed with the GENE INSPECTOR 1.5 program from Textco.
Laccase expression in T. versicolor.
Pre-cultures were prepared and 25 ml diluted into different 200 ml batches of expression medium containing 0, 100, 500, 1000, 2000 or 3000 µM CuSO4. These were grown at 24 °C with shaking at 110 r.p.m., and the cultures were induced on the second day with 1·5 mM 2,5-xylidine. From the third day onwards, laccase production was measured for 10 days using an ABTS test as described below. The experiment was performed in duplicate and the activity values were averaged between the duplicate cultures.
Measurement of laccase enzyme activity (ABTS test).
Laccase activity was determined in MacIllvain buffer pH 4·5 (0·1 M citrate with 0·2 M disodium hydrogen phosphate to the desired pH) by monitoring the oxidation of ABTS (Sigma) at 420 nm with a specific extinction coefficient of 3·6x104 l M-1 cm-1. The test was performed at 37 °C in 990-x µl MacIllvain buffer plus x µl enzyme and 10 µl of 10 mM ABTS. After incubation for 530 min, the reaction was stopped by adding 200 µl of 5 M NaCl. The volume activity was then calculated by c (U ml-1)=[E(1200/x)]/(36xt), where t is the time of incubation, x is the amount (µl) of enzyme used in the reaction and
E is the difference between the OD420 value of the ABTS test and that of the control (test without enzyme).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast transformants were isolated, further selected on Ferrozine medium and plasmids were isolated from selected clones for analysis (Methods). Of the 70 plasmids tested, 62 contained a 3 kb cDNA insertion (the others contained no insert), proving that complementation of the ccc2 mutant was very specific and probably involved one gene. To confirm this, a number of cDNA inserts from different clones were sequenced and a database search was performed on these sequences. All the clones showed clear homology to the known yeast, human and plant P-type copper ATPases Ccc2, Menkes and RAN1.
The cDNA sequences of clones 21 and 34 differ significantly from each other: comparison of their complete sequences revealed a total of 152 nt differences that result in 11 aa substitutions. To answer the question of whether these differences are due to allelic variations in ctaA or represent two different genes, Southern blot analysis was performed on restriction digests of diploid (dikaryon) or haploid (monokaryon) T. versicolor genomes. From the Southern blot analysis, it is clear that two fragments hybridize in the dikaryon TV1, while only one band is detected with the monokaryon F2-100 (data not shown). Therefore, clones 34 and 21 represent different alleles of ctaA, and are subsequently called ctaA-1 (cta34) and ctaA-2 (cta21).
To confirm that both ctaA-1 and ctaA-2 can complement the yeast ccc2 mutant, the plasmids containing the two cDNA clones were transformed back into Strain 3. The transformants were subjected to a drop titre test on low iron medium (1 mM Ferrozine), to which some iron had been added (150 µM) to maintain better growth of the wild-type. As negative and positive controls, Strain 3 and the corresponding wild-type strain YPH250 were transformed with the empty pAH vector and similarly subjected to the drop titre test. The
ccc2 Strain 3 clearly showed no growth on the selection plate (1 mM Ferrozine, 150 µM iron), while the strains transformed with the constructs pAHcta21 and pAHcta34 similar to the wild-type strain grew well (Fig. 1
). Thus, all the cDNAs cloned from T. versicolor show efficient complementation.
|
Characterization of ctaA
Comparison of the structural gene (deposited in GenBank under accession number AY210894) of allele ctaA-1 with the cDNA showed that the gene is interrupted by three introns between the start codon ATG and the double stop codons TGA TAG, whereby intron II, with 643 nt, is unusually long for a fungus. A fourth and alternatively spliced intron is found between the stop codons and the putative polyadenylation signals AATATT and ATAATT. Intron I is 50 nt long, intron III is 53 nt long and intron IV is 100 nt long. The consensus sequences for the splice sites correlated with those described for tahA and the fungi in general (Ballance, 1986). The isolated 2200 bp 5'-region of allele ctaA-2 stretches from the promoter region until shortly beyond intron II. Like intron II in allele ctaA-1, this intron is unusually large, although with 284 nt it is less than half the size of intron II in ctaA-1. However, analysis of both giant introns revealed no unusual sequence homology that might indicate, for example, an insertion element, and the sequence of the 5'- and 3'-splice sites of the intron correlated with those described for tahA and the fungi (Ballance, 1986
). Comparison of the promoter regions of both alleles showed that the first 430 nt upstream of the start codon (ATG) are highly conserved. Many sequences with homology to described elements that can play a role in transcription regulation were found in both alleles, including homologies to the binding sites for the transcription factors NIT2, GATA-1, PEA3, NF1, GCN4, GCR1 and Sp1. To what extent these elements influence the transcription of ctaA remains to be elucidated. A TATA box was not found, although a pyrimidine-rich sequence was detected directly before the start codon (ATG).
Characterization of the protein sequence of ctaA
The cDNA of ctaA encodes a protein of 983 aa, which shares up to 40 % identity and up to 60 % homology with other P-type copper ATPases. As with yeast Ccc2 and the human Menkes protein, eight transmembrane domains could also be predicted for CtaA since sequences in this region are highly conserved. Highly conserved motifs essential for the function of all copper-transporting P-type ATPases were also found in CtaA. For example, at positions 50 and 124 in the N terminus are two putative copper-binding sites with the peptide sequences GMTCGAC and GMTCSSC, which correspond to the consensus sequence GMTCxxC for copper-binding sites in proteins. A phosphatase site, TGEP, is found at position 407 and the APCPxLG motif important for copper transduction is located in membrane domain V at position 527, followed by a phosphorylation site, DKTGT (572), and an ATP-binding domain, GDGIND (816). The SEHPL motif, as yet not precisely characterized functionally, is found in CtaA at position 633. A methionine involved in copper transduction localized in membrane domain VIII is also conserved in CtaA. At the protein level the two CtaA alleles differ by 11 aa substitutions, none of which is located in any of these motifs.
Coexpression of tahA, ctaA and laccase in S. cerevisiae
To investigate the effect on laccase expression of deregulated expression of ctaA plus another copper-trafficking factor, tahA, which we identified previously (Uldschmid et al., 2002), the lacIII gene from T. versicolor was coexpressed with ctaA, tahA or ctaA plus tahA in the S. cerevisiae strain CM3260. In addition, strain CM3260 that already carries the laccase expression construct pLacP2 was transformed with different combinations of empty vectors (pAH and pAT) and expression constructs (pATtahA and pAHctaA: Methods, Fig. 2
). The wild-type yeast strain CM3620 used in this test shows no laccase activity at all (data not shown), since yeast do not have an endogenous laccase gene. As shown in Fig. 3
, overexpression of either tahA or ctaA had a positive effect on heterologous laccase expression in yeast. Overexpression of tahA showed the largest effect between 200 and 400 µM CuSO4, where up to three times as much laccase was produced compared to the parent strain. More dramatically, overexpression of ctaA resulted in an up to 15-fold increase in laccase expression compared to the control strain. Coexpression of ctaA and tahA showed an additive effect, particularly at very low copper concentrations (
10 µM), where up to 20-fold more laccase was expressed than in the control strain. Thus, overexpression of the T. versicolor genes tahA and ctaA can clearly increase the expression of lacIII in yeast.
A similar result was obtained using the yeast homologues of tahA and ctaA, ATX1 and CCC2 (Fig. 4) in an analogous experiment. The overexpression of either ATX1 or CCC2 also had a positive effect on laccase expression. An additive effect with the coexpression of ATX1 and CCC2 was also seen, since laccase activity could be increased twofold. However, with low copper concentrations, the effect of Atx1p and Ccc2p was not as strong as observed for the coexpression of the T. versicolor genes tahA and ctaA.
|
A more precise idea of the number of expression constructs integrated into the genome was obtained by isolating genomic DNA from the cotransformants Ko7, Ko15 and Ko19 and the recipient strain F6 and performing Southern blot analyses (Fig. 6). Hybridization of tahA in the recipient strain F6 showed only one cross-hybridizing band per digest, whereas the cotransformants had additional bands due to integration of the expression construct. The 1·5 kb band in the BamHI/NsiI digest corresponds to the complete tahA expression cassette with the gapDH promoter and tahA gene, since in the expression construct BamHI cuts before, and NsiI after, this cassette.
|
Laccase expression in T. versicolor cotransformants
In a preliminary test, the laccase expression of the T. versicolor cotransformants was compared to the wild-type F6 strain. For this purpose, strains Ko7, Ko15 and Ko19 and the control strain F6 were grown in liquid minimal medium without any additional copper. The laccase activity in the cultures was determined 7 days after induction with 2,5-xylidine. The cotransformants Ko7, Ko15 and Ko19 showed the highest laccase expression, ranging from fourfold for Ko15 (0·028 U ml-1), to fivefold for Ko19 (0·035 U ml-1), up to more than tenfold for Ko7 (0·092 U ml-1), compared to F6 (0·007 U ml-1).
Due to the promising result obtained with the Ko7 derivative, it was further characterized by analysing its laccase expression under different copper concentrations compared to the wild-type F6 strain. It was clear that all the Ko7 cultures supplemented with copper showed higher laccase expression than the F6 cultures. The difference was most obvious with 500 µM CuSO4, where the Ko7 derivative showed up to six times more laccase activity than the F6 strain. The Ko7 culture without added copper showed no elevated laccase production and resembled the F6 culture without copper. At 3000 µM copper F6 produced hardly any more laccase than in the control culture without copper, while Ko7 still produced it well (Fig. 7).
|
In conclusion, as in yeast, overexpression of the copper homeostasis factors tahA and ctaA leads to a significant increase in laccase expression.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 983 aa protein predicted from the cDNA sequence of ctaA showed up to 60 % homology to the known copper P-type ATPases Ccc2 from yeast and human MNKP (Menkes Disease Protein). This family of proteins is highly conserved in all organisms from all kingdoms. There are always one to six metal-binding sites (MBSs) at the N terminus (Pena et al., 1999) with the consensus sequence GMTCxxC. Like Ccc2, CtaA also has two MBSs at its N terminus. For Ccc2 it was shown that Atx1 binds to this motif and passes on copper (Pufahl et al., 1997
; Huffman & O'Halloran, 2000
). Since ctaA fully complements the functionality of the
ccc2 yeast mutant, the two MBSs of the CtaA protein probably also interact with Atx1 to take up the copper. Forbes et al. (1999)
could show by deletion analysis of the MBSs in WNDP (Wilson Disease Protein) that the MBS localized nearest to the first membrane domain is sufficient and also essential for copper transport. An additional highly conserved region is the CPC motif in membrane domain VI, which is essential for copper transduction. It could be shown that methionine 1386 in membrane domain VIII of the mouse WNDP is also essential (Voskoboinik et al., 2001
). This methionine is conserved in all copper P-type ATPases and is also present in CtaA. The ATP-binding domains, the phosphorylation domains and the dephosphorylation domains, where the turnover of ATP to ADP that drives copper transport takes place, were all identified in CtaA. From this, one can conclude that CtaA in T. versicolor performs the same function as Ccc2 in yeast and that it is localized in the Golgi network.
A more precise description of the function of CtaA in T. versicolor might be obtained from a ctaA knock-out strain. However, despite repeated attempts we have been unable to generate this so far. One reason for this could be that one of the five laccase isoenzymes from T. versicolor might be essential for cell development and/or morphogenesis. If this laccase can no longer be loaded with copper in a ctaA strain, this would mean that a knock-out strain could not grow. In addition, a hydroxylation step in the biosynthesis of ceramides in S. cerevisiae has been described as being dependent on functional Ccc2 (Beeler et al., 1997
; Haak et al., 1997
). A similar effect could also influence cell development in T. versicolor. In support of this theory is the fact that transformation of T. versicolor with a ctaA antisense construct produced very few transformants. Antisense RNA expression may lead to the same lethal result as a potential ctaA knock-out. This question could be answered in the future by more detailed analysis of an antisense construct or by using RNA interference (RNAi).
Several recognition motifs for transcription factors such as NIT2, GCN4 and NF1 were found in T. versicolor, although a transcript could not be detected by Northern blotting (not shown). It remains unclear whether the very large second intron plays a role in the very low transcription level of ctaA, although it is possible the length of the intron could have an indirect influence on the number of transcripts via splicing activity (Pukkila & Casselton, 1991). A second possibility could be that the intron forms a kind of terminator structure and thus full-length ctaA transcripts are kept at a low level. The transcriptional regulation of homologous proteins has yet to be investigated in any detail. For example, four metal response elements (MREs) detected in the promoter of the WND gene (Oh et al., 2002
) have not yet been tested for copper induction. The P-type ATPases can probably be considered household genes that are often regulated at the protein level. However, to date, it could only be shown for the homologous copper P-type ATPases WNDP and MNKP that their localization changed according to copper concentrations. At low copper concentrations they are found in the Golgi and at high copper concentrations they are localized at the cell membrane where they pump copper out of the cells (Petris et al., 1996
; Hung et al., 1997
).
Coexpression of tahA and ctaA in yeast and in T. versicolor
We show here that deregulated coexpression of ctaA and tahA in a laccase III-expressing yeast could lead to increased laccase yields by up to 20-fold with a copper concentration of 10 µM. Analogous results were also obtained by overexpressing ATX1 and CCC2, the yeast homologues of tahA and ctaA. However, although the increase in laccase expression was not as high as that mediated by tahA and ctaA coexpression, it was nevertheless significant. We then determined that the deregulated coexpression of both these genes in T. versicolor also led to an increase in laccase expression of up to eightfold. This shows that the efficiency of laccase production depends on the supply of copper to the Golgi vesicle and that overexpression of the genes that supply this cell organelle with copper apparently increase the copper flow in this direction. Since overexpression of ctaA in yeast under low copper concentrations showed the more dramatic effect on laccase expression, and CtaA is probably regulated more at the protein level, one might suppose that the CtaA protein is the bottleneck in copper transport to the Golgi lumen. Further investigations into the regulation of CtaA protein activity are needed to determine to what extent CtaA function is influenced post-translationally, for example, by phosphorylation. Examples described by Shatzman & Kosman (1978), analysing galactose oxidase, Cu/Zn-SOD and cytochrome c oxidase, showed that under limiting copper conditions the copper supply is first regulated in secreted proteins, then in Cu/Zn-SOD, followed by the respiratory chain proteins. In T. versicolor under copper-deficient conditions, the copper supply to the Golgi is minimized by down-regulation of TahA and laccase is no longer supplied with sufficient copper. The fungus, however, grows quite normally under these conditions, which shows that its respiratory chain proteins still receive copper. The overexpression of both genes (ctaA and tahA) probably results in a strong interference in this hierarchy of copper distribution to the advantage of the Golgi network. A kind of copper highway is achieved that directs the major part of the available copper pool to the Golgi. However, this has no effect on the growth of the fungus, which shows that an undersupply is not created for the important respiratory copper enzymes such as COX (cytochrome c oxidase). The increased copper transport to the Golgi described above could also be the reason why the cotransformant Ko7 still expresses laccase at 3000 µM copper while hardly any laccase activity is found in the wild-type F6 strain. Under these conditions all the copper systems are probably repressed in F6 due to the high amounts of copper, while all the copper detoxification proteins such as metallothionein, as well as copper chaperones and Cu/Zn-SOD, are highly expressed. The expression of phytochelatins under copper stress has been shown for Schizosaccharomyces pombe (Perego et al., 1996
). Whether, and in what way, these mechanisms apply to T. versicolor must be elucidated by future experiments.
Since metallothionein and phytochelatin have a very high affinity for copper (e.g. >2x1014 M-1 for human MT-3) (Hasler et al., 2000), they probably compete with the copper chaperone TahA for the available copper. This would undermine the supply of copper to the Golgi and laccase would no longer be supplied with copper. In Ko7 the overexpression of TahA and CtaA may relieve the competition with metallothioneins, meaning laccase is still supplied with copper. This might prove an important consideration if, for example, one was constructing laccase-overproducing strains for biotechnological purposes.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartholomew, K., Dos Santos, G., Dumonceaux, T., Charles, T. & Archibald, F. (2000). Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble. Appl Microbiol Biotechnol 56, 201204.
Beeler, T. J., Fu, D., Rivera, J., Monaghan, E., Gable, K. & Dunn, T. M. (1997). SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 °C, is required for mannosylation of inositolphosphorylceramide. Mol Gen Genet 255, 570579.[CrossRef][Medline]
Call, H. P. & Mücke, I. (1997). History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozyme®-process). J Biotechnol 53, 163202.[CrossRef]
Eide, D. J., Bridgham, J. T., Zhao, Z. & Mattoon, J. R. (1993). The vacuolar H(+)-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism. Mol Gen Genet 241, 447456.[Medline]
Felby, C., Petersen, L. S. & Nielsen, B. R. (1997). Enhanced auto adhesion of wood fibers using phenol oxidases. Holzforschung 51, 281286.
Feldmann, H., Driller, L., Meier, B., Mages, G., Kellermann, J. & Winnacker, E. L. (1996). HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae. J Biol Chem 271, 2776527769.
Forbes, J. R., Hsi, G. & Cox, D. W. (1999). Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease. J Biol Chem 274, 1240812413.
Froeliger, E. H., Ullrich, R. C. & Novotny, C. P. (1989). Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune. Gene 83, 387393.[Medline]
Gurr, S. J., Unkles, S. E. & Kinghorn, J. E. (1988). The structure and organization of nuclear genes of filamentous fungi. In Gene Structure in Eucaryotic Microbes, pp. 93139. Edited by J. E. Kinghorn. Oxford: IRL Press.
Haak, D., Gable, K., Beeler, T. & Dunn, T. (1997). Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p. J Biol Chem 272, 2970429710.
Hasler, D. W., Jensen, L. T., Zerbe, O., Winge, D. R. & Vasak, M. (2000). Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein. Biochemistry 39, 1456714575.[CrossRef][Medline]
Himelblau, E., Mira, H., Lin, S. J., Culotta, V. C., Penarrubia, L. & Amasino, R. M. (1998). Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis. Plant Physiol 117, 12271234.
Hirayama, T., Kieber, J. J. & Hirayama, N. & 7 other authors (1999). RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis. Cell 97, 383393.[Medline]
Huffman, D. L. & O'Halloran, T. V. (2000). Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2. J Biol Chem 275, 1861118614.
Hung, I. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D. & Gitlin, J. D. (1997). Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae. J Biol Chem 272, 2146121466.
Klomp, L. W., Lin, S. J., Yuan, D. S., Klausner, R. D., Culotta, V. C. & Gitlin, J. D. (1997). Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis. J Biol Chem 272, 92219226.
Logemann, J., Schell, J. & Willmitzer, L. (1987). Improved method for the isolation of RNA from plant tissues. Anal Biochem 163, 1620.[Medline]
O'Halloran, T. V. & Culotta, V. C. (2000). Metallochaperones, an intracellular shuttle service for metal ions. J Biol Chem 275, 2505725060.
Oh, W. J., Kim, E. K., Ko, J. H., Yoo, S. H., Hahn, S. H. & Yoo, O. J. (2002). Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene. Eur J Biochem 269, 21512161.
Payne, A. S. & Gitlin, J. D. (1998). Functional expression of the Menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases. J Biol Chem 273, 37653770.
Pena, M. M., Lee, J. & Thiele, D. J. (1999). A delicate balance: homeostatic control of copper uptake and distribution. J Nutr 129, 12511260.
Perego, P., Jimenez, G. & Howell, S. B. (1996). Isolation and characterization of a cisplatin-resistant strain of Schizosaccharomyces pombe. Mol Pharmacol 50, 10801086.[Abstract]
Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A. & Camakaris, J. (1996). Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J 15, 60846095.[Abstract]
Pufahl, R. A., Singer, C. P., Peariso, K. L., Lin, S. J., Schmidt, P. J., Fahrni, C. J., Culotta, V. C., Penner-Hahn, J. E. & O'Halloran, T. V. (1997). Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science 278, 853856.
Pukkila, P. J. & Casselton, L. A. (1991). Molecular genetics of the agaric Coprinus cinerius. In More Gene Manipulations in Fungi, pp. 126150. Edited by J. W. Bennett & L. L. Lasure. San Diego: Academic Press.
Rosenau, T., Potthast, A., Chen, C. L. & Gratzl, J. S. (1996). A mild, simple and general procedure for the oxidation of benzyl alcohols to benzaldehydes. Synth Commun 26, 315320.
Sariaslani, F. S. (1989). Microbial enzymes for oxidation of organic molecules. Crit Rev Biotechnol 9, 171257.[Medline]
Shatzman, A. R. & Kosman, D. J. (1978). The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides. Biochim Biophys Acta 544, 163179.[Medline]
Uldschmid, A., Engel, M., Dombi, R. & Marbach, K. (2002). Identification and functional expression of tahA, a filamentous fungal gene involved in copper trafficking to the secretory pathway in Trametes versicolor. Microbiology 148, 40494058.
Voskoboinik, I., Greenough, M., La Fontaine, S., Mercer, J. F. & Camakaris, J. (2001). Functional studies on the Wilson copper P-type ATPase and toxic milk mouse mutant. Biochem Biophys Res Commun 281, 966970.[CrossRef][Medline]
Wakabayashi, T., Nakamura, N., Sambongi, Y., Wada, Y., Oka, T. & Futai, M. (1998). Identification of the copper chaperone, CUC-1, in Caenorhabditis elegans: tissue specific coexpression with the copper transporting ATPase, CUA-1. FEBS Lett 440, 141146.[CrossRef][Medline]
Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T. & Klausner, R. D. (1995). The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake. Proc Natl Acad Sci U S A 92, 26322636.[Abstract]
Received 9 December 2002;
revised 7 April 2003;
accepted 28 April 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |