Metal transportome of Neurospora crassa

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Abstract

Neurospora crassa has been the model filamentous fungus for the study of many fundamental cellular mechanisms of transport and metabolism. The recently completed genome sequence of N. crassa has over 10,000 genes without significant matches for a large number of genes (41%) in the sequence databases, indeed presents many challenges for new discoveries. Using transporter database and BLAST searches a total of 65 open reading frames for putative cation transporter genes have been identified in N. crassa. These were further confirmed by characteristic features of the family like transmembrane domains (TOPPRED 2), conserved motifs (Clustal W) and phylogenetic analysis (TREETOP). In Neurospora cation transporter genes constitute nearly 18.3% of the total membrane transport systems, which is higher than E. coli (8.8%), S. cerevisiae (13.7%), S. pombe (17.2%), A. fumigatus (10.1%), A. thaliana (16.8%) and H. sapiens (15.6%). We refer to the complete complement of metal ion transporter genes as "Metal Transportome". There are a total of 33 putative transporters for alkali and alkaline earth metals constituting 18 for calcium (P-ATPase, VIC, CaCA, Mid1), 7 for sodium (P-ATPase, CPA1, CPA2), 4 for potassium (Trk, VIC, KUP), and 4 for magnesium (MIT). Transition metal ion transporters account for 32 transporters including 7 for zinc (ZIP), 6 for copper (Ctr2, Ctr1), 2 each for manganese (Nramp), iron (OFeT), arsenite (ArsAB, ACR3) and other metal ions (ABC and P-ATPase) and 1 each for nickel (NiCoT) and chromate (CHR). N. crassa has 7 linkage groups of which LGI harbors 21 of metal ion transporters and in contrast LGVII has only 2. Studies on metal transportomes of different organisms will help to unravel the role of metal ion transporters in homeostasis.

Keywords: metal ion transporters, transportome, metals, transporters, Neurospora crassa

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Introduction

Neurospora crassa is a multicellular filamentous fungus playing central role as model organism in the history of twentieth century genetics, biochemistry and molecular biology. N. crassa was developed as an experimental organism in 1920s [Shear, 1927; Lindegren, 1936]. Subsequent work on Neurospora by Beadle and Tatum [Beadle et al., 1941] in the 1940s established the relationship between genes and proteins summarized in the 'one gene - one enzyme' hypothesis. In the later half of the century studies using Neurospora contributed to the fundamental understanding of genome defense systems, DNA methylation, circadian rhythms and DNA repair. Recently N. crassa gained additional significance due to sequencing of the whole genome [Galagan et al., 2003], which opened up new questions. Metal ions are required both in bulk or trace quantities for growth and optimal metabolism. However some of the essential trace metal ions are toxic at relatively higher concentrations. Hence their homeostasis is crucial for all living organisms. Metal ion transporters play a vital role to face competition in limited resources and at the same time their regulation provides solution to the changing environment and the potential damage caused by abnormal concentrations. Cells need to maintain certain cytoplasmic concentrations of these metals to meet the physiological requirements. Although an excess of metals is generally toxic, some of the metals (Ca, Mg, K, Na, Mn, Fe, Zn, Cu, Co, Ni, and Cd) are essential to life for optimal growth, metabolism, development and reproduction. The Transport Classification (TC) system [Saier, 2000] represents a systematic approach to organize transport systems according to the mode of transport, energy-coupling mechanism, molecular phylogeny and substrate specificity. Based on TC system, Transport database- a relational database is designed for describing the cellular membrane transport proteins in organisms whose complete genome sequences are available [Ren et al., 2004]. This paper describes identification of genes predicted to be involved in metal ion transport of N. crassa.

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Methods

The complete sequence of the genome of Neurospora crassa is available at the FGSC site (http://www.fgsc.net/). For the identification of proteins the BLAST program [Altschul et al., 1990] was used with functionally characterized proteins of other organisms. ORFs identified in the N. crassa genome were BLAST searched back against the Swiss-Prot/TrEMBL database and only functionally characterized proteins in the database were taken into account for assigning predicted role to the proteins encoded by genes in the N. crassa genome. All the metal transporting genes were classified according to transport database (http://www.membranetransport.org/). The transmembrane domains were obtained using TOPPRED (http:// bioweb.pasteur.fr/seqanal/interfaces/toppred.html) software based on Kyte-Doolittle scale of hydrophobicity [Kyte and Doolittle, 1982] and Positive-inside rule. The conserved residues were analyzed by multiple sequence alignment using CLUSTALW (http://www.ebi.ac.uk/clustalw) software. Phylogenetic analysis was performed using TREETOP software (http://www.genebee.msu.su/services/phtree_reduced.html) and trees were bootstrapped 100 times to ensure the reliability of each branch point [Yushmanov and Chumakov, 1988].

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Results and discussion

Basic features of metal transportome

Computer-assisted BLAST searches and transport database search of the Neurospora crassa genome identified a number of putative genes involved in the uptake and extrusion of metals. A total of 65 open reading frames with a putative role in metal homeostasis are identified and summarized in Table 1. Comparative evaluation of metal ion transportomes of some representative organisms shows the presence of more percent of cation transporters in N. crassa, which account for nearly 18.3% of total membrane transporter genes as shown in Fig. 1. E. coli has the least (8.8%) followed by A. fumigatus (10.1%), S. cerevisiae (13.7%) and H. sapiens (15.6%) while A. thaliana (16.8%) and S. pombe (17.2%) are closer to N. crassa. All the members are placed in their respective families according to the TC system of classification based on characteristic features like transmembrane domains, hydropathic plots, conserved motifs/ residues and molecular phylogeny.

Alkali and alkaline earth metal transporters

Alkaline earth metals (Na, K, Ca and Mg) are essential for all living organisms and maintained by fine homeostatic control mechanisms involving pumps and channels. Among these, Ca²⁺ and Na⁺ P-type ATPases play a central role. P-type ATPases make up a large superfamily of ATP-driven pumps, which share a common structure and a common mechanism of activity. In this superfamily, Ca²⁺-, Na⁺/K⁺-, H⁺/K⁺-, K⁺/H⁺- and fungal Na⁺-ATPases form a subgroup, named type II [Møller et al., 1996; Axelsen and Palmgren, 1998]. Some of the Na⁺ and Ca²⁺ ATPases of N. crassa (NCU05046.2, NCU03292.2, NCU03305.2, NCU04736.2, NCU08147.2 and NCU05154.2), which respond to stress conditions, have already been characterized [Benito et al., 2000]. There are four other members in P-type ATPases that are predicted to be involved in cation transport. A detailed in silico analysis of N. crassa genome sequence in which putative genes involved in calcium signaling has been discussed earlier [Borkovich et al., 2004; Zelter et al., 2004]. These include all the members involved in calcium transport (Table 1).

Na⁺/H⁺ exchangers (NHE) are a ubiquitous family of transmembrane proteins that catalyze the antiport of Na⁺ and H⁺ at the plasma membrane. Under physiological conditions, NHE drive H⁺ out of the cell by coupling to the Na⁺ gradient, which is generated and maintained by the activity of the Na⁺/K⁺-ATPase. NHE are implicated in a variety of important physiological functions, including intracellular pH regulation, cell volume control, and Na⁺ homeostasis. NHEs of N. crassa are divided into 2 sub-families, CPA1 (Cation:proton antiporter) and CPA2. Hydropathy analysis of the NHE isoforms of N. crassa predicted to have 10 to 12 transmembrane helices in the N-terminus and that the C-terminus constitutes a large cytoplasmic domain. Neurospora has six Ca²⁺/H⁺ exchangers also of which one, i. e. CAX (NCU07075.2), was already characterized [Margolles-Clark, 1999].

Potassium is the nutrient maintained at the highest concentration in the cells and one of the nutrients that requires being transported against the highest transmembrane concentration gradients when the external medium is dilute. There are four K⁺ transporters in N. crassa of which two (NCU06449.2 and NCU00790.2) are already characterized [Haro et al., 1999], one belongs to TRK family [Gaber et al., 1988; Bertl et al., 2003; Nakamura et al., 1998] and the other belongs to VIC super family [Maingret et al., 1999]. These proteins possess 8 putative transmembrane α-helical spanners. An 8 TMS topology with N- and C-termini on the inside has been established for AtHKT1 of A. thaliana [Kato et al., 2001] and Trk2 of S. cerevisiae [Zeng et al., 2004].

Mg²⁺ is the most abundant divalent cation in cells. It is essential for the activation of hundreds of enzymes, for the maintenance of active conformations of macromolecules, for charge compensation, and for the modification of various ion channels. In bacteria, three proteins (CorA, MgtA, and MgtB) have been shown to be involved in Mg²⁺ transport across the plasma membrane [Smith, 1998]. In N. crassa four putative genes belonging to CorA family are identified. They are characterized by two or three adjacent transmembrane domains near their carboxyl termini, one of which is followed by the (Y/F/W)GMN motif.

The ABC superfamily contains both uptake and efflux transport systems, and the members of these two porter groups generally cluster loosely together with just a few exceptions. ATP hydrolysis without protein phosphorylation energizes transport. ABC-type uptake systems have not been identified in eukaryotes, but ABC-type efflux systems are commonly found in both prokaryotes and eukaryotes. The eukaryotic efflux systems often have four domains (two cytoplasmic domains and two integral membrane domains) fused into either one or two polypeptide chains. The integral membrane porter domains each usually possess 5 (uptake) or 6 (efflux) transmembrane spanners, but exceptions exist. For example, the MntB protein (TC #3.A.1.15.1) exhibits 9 established TMSs [Schmitt and Tampé, 2002]. N. crassa has a single member belonging to this family involved in metal ion efflux consisting of ten transmembrane domains.

Transition metal ion transporters

Transition metals are essential for function of many proteins, either by facilitating redox reactions or by stabilizing the protein structure. These transition metal transport systems consists of both high and low affinity transporters to maintain the homeostasis in either metal limiting or replete conditions. The Neurospora homologues of transition metal transporter proteins involved in metal transport are described in Table 2.

The CDF (Cation Diffusion Facilitator) family is ubiquitous family comprising members represented at all phylogenetic levels transporting metals like cobalt, cadmium, zinc and nickel. N. crassa has nine members in this family and they possess five or six putative transmembrane spanners characteristic to this family and size ranges from 300-750 amino acid residues. Eukaryotic proteins exhibit differences in cell localization and are found in plasma membranes and organellar membranes [Chao and Fu, 2004; Haney et al., 2005; MacDiarmid et al., 2003].

The Zrt-Irt like protein (ZIP) family members are involved in Zn²⁺/Fe²⁺uptake named after the first identified members Zrt1 of S. cerevisiae [Zhao and Eide, 1996a] and Irt1 of A. thaliana [Eide et al., 1996]. N. crassa consists of 7 members belonging to this family that are involved in zinc uptake. Members of the ZIP family consist eight putative transmembrane spanners, amino and carboxy termini are cytoplasmic, long loop region between transmembrane domains III and IV and transmembrane domains IV and V contain conserved histidine residues characteristic to this family. All these features are present in most of the N. crassa members. Phylogenetic analysis as showed in Fig. 2a predicted one member homologous to Zrt1 and one to Zrt2 [Zhao and Eide, 1996b] indicating the presence of high and low affinity transporters involved in zinc uptake.

Nickel is an essential component of at least nine metalloenzymes involved in energy and nitrogen metabolism. Synthesis of nickel is dependent on high-affinity uptake of this metal ion from natural environments where they are available only in trace amounts. NiCoT, a family of secondary transporters in prokaryotes and fungi mediate high-affinity uptake of nickel or cobalt ions into the cells. N. crassa has a single member belonging to this family with 7 transmembrane domains and a conserved signature sequence (RHALDADHI) that is the characteristic feature of NiCoT family. Multiple sequence alignment showed N. crassa member to be a closer homologue to HoxN, a high-affinity nickel uptake permease of Ralstonia eutropha [Degen and Eitinger, 2002; Eitinger et al., 2005 ]. However till date the importance of either Ni²⁺ or Co²⁺ in N. crassa metabolism has not been characterized.

Nramp (Natural resistance-associated macrophage proteins) family found in mammals, birds, nematodes and insects. It is hypothesized that a deficiency for Mn²⁺ or some other metal prevents the generation of reactive oxygenic and nitrogenic compounds that are used by macrophages to combat pathogens. N. crassa has two members that are predicted to be involved in manganese transport. They have 11 transmembrane domains similar to that characterized in E. coli [Courville et al., 2004].

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, oxidative stress protection, hormone production and pigmentation. However, with excess accumulation copper generates hydroxyl radicals that damage cells at the level of nucleic acids, proteins and lipids. A widely conserved family of high affinity copper transport proteins (Ctr family) mediates copper uptake at the plasma membrane [Zhou and Thiele, 2001; Zhou and Gitschier, 1997]. N. crassa has three members (NCU00830.2, NCU03281.2 and NCU07428.2) belonging to this family. Ctr family members contain two or three membrane-spanning domains and a G4 motif that helps in multimerization of copper monomers, localization of multimer and copper transport [Aller et al., 2004]. This G4 motif is conserved in N. crassa members also.

Distinct bacterial enzymes specific for K⁺ or Mg²⁺ (uptake), Ca²⁺, Ag⁺, Zn²⁺, Co²⁺, Pb²⁺, Ni²⁺, and/or Cd²⁺ (efflux) and Cu²⁺ or Cu⁺ (uptake or efflux, depending on the system) have been characterized, and each of these enzymes comprises a distinct subfamily. Cu²⁺ or Cu⁺-translocating ATPases from bacteria, archaea and animals cluster together, and at least some of these also transport Ag⁺. The Cu⁺/Ag⁺ ATPases have an 8 TMS topology [Mandal et al., 2002]. N. crassa has four hypothetical proteins that are predicted to translocate Cu²⁺ driven by ATP hydrolysis, which has same 8 TMS topology. A Cys-Pro-Cys motif in CopA of E. coli is essential for Cu⁺/Ag⁺ efflux and phosphoenzyme formation [Fan and Rosen, 2002] and this motif is present in three members of N. crassa ATPases (NCU04076.2, NCU07531.2 and NCU08341.2). N. crassa has two highly similar putative copper transporters (NCU07531.2 and NCU08341.2) associated with Wilson's disease, an inherited disorder that causes the body to retain copper and can result in severe brain damage, liver failure and death.

Iron is the most versatile transition metal ion in biological redox reactions. High affinity iron transport is mediated by a bipartite system composed of a ferrooxidase and transmembrane permease. The ferrooxidase Fet3p is a multicopper oxidase that catalyzes the oxidation of Fe(II) with the concomitant reduction of molecular oxygen [Askwith, 1994]. The oxidized Fe(III) is then transported by a multitopic membrane protein encoded by the Ftr1 gene [Stearman,1996]. These two come under OFeT (oxidase-dependent iron transporter) family, which consists of high affinity iron transporters. N. crassa has two members of which NCU03498.2 resembles Fet3 and NCU03497.2 resembles Ftr1 as shown in Fig. 2b. The later member has six transmembrane domains and an iron binding motif with conserved residues REGVE [Stearman,1996] as in case of Ftr1. Co-expression studies of both the iron transport systems either in S. cerevisiae and S. pombe shows that it is sufficient to confer high-affinity iron transport [Askwith and Kaplan, 1997]. These similarities suggest the presence of bipartite system for iron transport in N. crassa and wet lab work will help in understanding the transport process of this essential trace element.

Toxic ion uptake

We are referring to toxic metal ions herein as those which produce toxicity at relatively low concentrations and are not reported to be useful for any organism. Toxicity could result in binding to essential respiratory chain proteins, oxidative damage via the production of reactive oxygen species, inhibition of enzyme activity, antagonisms or DNA damage. N. crassa has three members likely to be involved in uptake of chromate and arsenite. CHR proteins occur in bacteria and archaea. They consist of about 400 amino acid residues, appear to have 10 transmembrane-helical segments in an unusual 4+6 arrangement, and arose by an intragenic duplication event. One member belongs to CHR (Chromate Ion Transporter) family with 9 putative transmembrane domains and two conserved motifs (GGX₁₂VX₄WX₁₆PGPX₁₀GX₇G [X = any residue] and GGX₁₂VX₄WX₁₆PGPX₈GX₇G) characteristic to this family [Nies et al., 1998]. Two members are involved in arsenite uptake one belonging to ArsAB (Arsenite-Antimonite efflux) family and the other to ACR3 (Arsenical Resistance-3) family. The arsenite resistance (Ars) efflux pumps of bacteria consist either of two proteins (ArsB, the integral membrane constituent with twelve transmembrane spanners, and ArsA, the ATP-hydrolyzing, transport energizing subunit). ArsA proteins have two ATP binding domains and probably arose by a tandem gene duplication event. Two proteins of the ACR3 family have been functionally characterized. These proteins are the ACR3 protein of Saccharomyces cerevisiae, also called the ARR3 protein [Wysocki et al., 1997], and the "ArsB" protein of Bacillus subtilis [Sato and Kobayashi, 1998]. One (NCU06717.2) is closer homologue to ArsA protein and other (NCU01852.2) to ACR3 protein of S. cerevisiae.

Linkage groups

Neurospora has seven chromosomes with well-defined genetic map and designated as LGI through LGVII. CHEF (Contour-Clamped Homogeneous Electric Fields) gel analysis shows that the chromosomes range between 4Mb (corresponding to linkage Groups VI and VII) and 10.3Mb (l

inkage group I) of DNA [Orbach et al., 1988]. Based on the contig and supercontig numbering we placed all the metal transporters to their respective linkage groups. The LGI harbors 21 of the metal ion transporters and in contrast LGVII harbor only 2 of them. Fig. 3 shows number of metal ion transporters in each linkage group.

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Concluding remarks

The availability of genome sequence of Neurospora crassa helped in the primary identification of putative metal transporter genes likely to be involved in metal homeostasis that is crucial for life. The most striking difference in N. crassa with other organisms is the presence of more percent of metal ion transporters than E. coli, S. cerevisiae, S. pombe, A. fumigatus, A. thaliana and H. sapiens. The presence of three new Ca²⁺ channel proteins, eight P-type Ca²⁺ ATPases, six Ca²⁺/H⁺ exchangers and two Ca²⁺/Na⁺ exchangers helps in better understanding of calcium transport and mechanism underlying the calcium homeostasis. The finding of two proteins related to Wilson's disease in N. crassa suggests the potential for studying subtleties in the copper uptake process in this organism. Apart from high and low affinity transporters for zinc there are five other transport systems predicted to be involved in zinc transport and a further study would help in better understanding of the mechanism of zinc homeostasis and resistance at orgenellar level. Further wet lab work needs to be carried out to identify the native substrates of the transporters described in this paper. Knockout mutants of Neurospora crassa would be useful in characterization of metal ion transporters and the recent Neurospora genome project initiative directed towards the above is in active progress (http://www.dartmouth.edu/~neurosporagenome/1_s1.html). Our laboratory continues to work on the above aspects in Neurospora crassa.

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Acknowledgements

The work was supported by the financial assistance from Department of Science and Technology (DST No: SP/SO/D-55/99) and UGC-SAP.