Fungal Genetics and Biology 66 (2014) 69–78

Contents lists available at ScienceDirect

Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi

Regular Articles

Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis Corinna Probst a,1, Phillip Ringel a,1, Verena Boysen b, Lisette Wirsing b, Mariko Matsuda Alexander a,2, Ralf R. Mendel a,⇑, Tobias Kruse a a b

Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany Cellular Proteomics, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany

a r t i c l e

i n f o

Article history: Received 22 November 2013 Accepted 14 February 2014 Available online 23 February 2014 Keywords: Neurospora crassa nit Mutant Molybdenum cofactor Molybdenum cofactor biosynthesis

a b s t r a c t Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Neurospora crassa (N. crassa) is a filamentous fungus which is commonly used as a model organism in genetics and other biological research fields, including the biology of cell morphogenesis and differentiation (Galagan et al., 2003; Borkovich et al., 2004). Importantly, findings from N. crassa can be translated to other eukaryotes as well, allowing the study of biological problems originally discovered in complex eukaryotic organisms (e.g. plants, humans). Research fields that benefit from N. crassa as model system are, for instance, related to neurobiology (Seiler et al., 1999; Seiler and Plamann, 2003) or cell communication and cell fusion (Fleißner, 2012). However, open questions about molybdenum (Mo)3 metabolism can also be addressed using N. crassa as a model system

⇑ Corresponding author. Address: Department of Plant Biology, Braunschweig University of Technology, Humboldt Street 1, 38106 Braunschweig, Germany. Fax: +49 531 3918128. E-mail address: [email protected] (R.R. Mendel). 1 These authors equally contributed to this work and are listed in alphabetical order. 2 Present address: Cornell University Ithaca, NY 14853, United States. 3 The abbreviations used are: Molybdenum (Mo), molybdopterin (MPT), molybdenum cofactor (Moco), cyclic pyranopterin monophosphate (cPMP), Moco dependent enzyme (Mo-enzyme), nitrate reductase (NR), nitrate non-utilizing (nit). http://dx.doi.org/10.1016/j.fgb.2014.02.004 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

(Ringel et al., 2013). Mo is a transition element, which – in order to gain biological activity within the cell – must be complexed with a low molecular weight compound. This scaffolding compound is a pterin derivative which in its empty form is called molybdopterin (MPT), and in its metal bound form is called molybdenum cofactor (Moco, for structures see Fig. 1, Mendel and Kruse, 2012; Mendel, 2013). Moco, and its biosynthesis pathway, is evolutionarily conserved among all eukaryotes. Notably, elucidation of the four-step pathway has been carried out first and foremost in bacteria (Rajagopalan and Johnson, 1992) and plants (Mendel, 2013) although it was originally identified in the fungus Aspergillus (Emericella) nidulans (A. nidulans) (Cove and Pateman, 1963; Pateman et al., 1964). Results from work with the model plant Arabidopsis thaliana revealed the mitochondrial localization of the first step of Moco biosynthesis (Teschner et al., 2010), where starting from GTP, cyclic pyranopterin monophosphate (cPMP, formerly known as precursor Z) is formed (Wuebbens and Rajagopalan, 1993; Rieder et al., 1998). Following mitochondrial export of cPMP into the cytoplasm, it is converted to MPT, the second detectable Moco intermediate (Mendel and Kruse, 2012). In the third step of Moco biosynthesis, MPT is adenylated, yielding MPT-AMP (Fig. 1, Kuper et al., 2004; Llamas et al., 2004). Further processing of MPT-AMP finally results in the formation of physiologically active Moco (fourth step, Llamas et al., 2006). Steps two to four of Moco

70

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

Fig. 1. The N. crassa molybdenum cofactor (Moco) biosynthesis. Neurospora crassa (N. crassa) molybdenum cofactor (Moco) biosynthesis genes are illustrated; exons are boxed in gray and abbreviated as E, introns are boxed in white, with the position of the introns within the gene given under the respective illustrations. From each Moco biosynthesis variant the wildtype gene and all known UV-mutants are shown. Functionally relevant residues are indicated by an asterisk (*), point mutations are indicated by an ( ), frame shift mutations are indicated with ( ). For nit-9B missing expression is indicated with an (X). All Moco biosynthesis intermediates are shown within the figure.

71

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

biosynthesis were shown to be localized in the plant cytoplasm, and research suggests that the participating enzymes form a multi-enzyme complex (Kaufholdt et al., 2013). The careful biochemical and cell biological characterization of this multi-step pathway recently permitted the first drug-based therapy for human Moco deficiency (Schwarz et al., 2004; Veldman et al., 2010), a rare and previously untreatable lethal genetic disorder. Moco is the essential part of all Moco-dependent enzymes (Mo-enzymes), except bacterial nitrogenase, where Mo is complexed in the iron-molybdenum cofactor (Hu and Ribbe, 2011; Seefeldt et al., 2009). In contrast to the well characterized Moco biosynthesis pathway, cellular Moco allocation (Kruse et al., 2010) and subsequent insertion into the various apo-Moenzymes is poorly characterized. Preliminary experiments looking at the principles behind Moco insertion have been carried out, using the apo-nitrate reductase (NR) from N. crassa as a model system (Ringel et al., 2013). Although characterized with detail in vitro, the cellular basis of Moco delivery to NR and the existence of interacting proteins possibly involved have not yet been revealed. In order to further characterize the in vivo maturation process of NR, fully defined N. crassa Moco biosynthesis mutant strains are necessary, allowing comparison between Mocodependent and Moco-independent NR interactions. There is limited information available about N. crassa Moco biosynthesis and biosynthesis-related mutants. Previous studies have performed biochemical characterization on mutants in the first, second, and third (fourth) steps of Moco biosynthesis; however, any genetic characterization other than mapping data is missing (Coddington, 1976; Tomsett and Garrett, 1980; Dunn-Coleman, 1984; Heck and Ninnemann, 1995). Furthermore, sequencing data revealing the nature of the underlying mutations is not available, thus the molecular reason for Moco deficiency in these strains is unknown. To complete the description of these mutants and to enable comparison with the corresponding system in A. nidulans, their careful genetic characterization is required. In this work, we review the preliminary annotated N. crassa Moco biosynthesis data and reveal a gene not previously identified in N. crassa. Additionally, we describe fullydefined Moco biosynthesis knock-out strains, which will be a valuable tool for characterization of the in vivo maturation process of the N. crassa NR. 2. Material and methods 2.1. Identification of N. crassa Moco biosynthesis genes Identification of N. crassa Moco biosynthesis gene loci was achieved by BLAST (Basic Local Alignment Search Tool, Altschul et al., 1990) searches using the protein sequences of the A. nidulans Moco biosynthesis enzymes as queries: CnxABC, GenBank: AAB84030.1; CnxF, GenBank: AAC24520.1; CnxG, GenBank: AAD39470.1; CnxH, GenBank: AAD39471.1; CnxE GenBank: AAK83300.1. 2.2. N. crassa Moco strains used in this study Strains used in this study are listed in Table 1. 2.3. N. crassa media and growth conditions Strains were grown on Vogel’s minimal medium (MM) slants (Vogel, 1956). For strains carrying resistance markers, required supplements were added to the medium. For initial screening of N. crassa Moco-depleted mutant strains, MM supplemented with sodium chlorate (300 mM) was used. MM without a reduced

Table 1 Strain origin and genotype. FGSC = Fungal Genetics Stock Center, nit = nitrate nonutilizing. Strain (strain number)

Genotype (allele)

Origin

FGSC 987 FGSC 9719 FGSC 32 FGSC 3933 FGSC 4236 FGSC 3935 FGSC 3936 FGSC 3937 NCU00498_ko NCU00736_ko NCU08375_ko NCU03170_ko NCU09746_ko

mat mat mat mat mat mat mat mat mat mat mat mat mat

FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC This study This study This study This study This study

(G253) (G117) (G179) (G123) (G128)

A (74-OR23-1A) a, delta mus-52::bar+ a, nit-1 (34547) A, nit-7 (V1M59) A, nit-8 (V1M44) A, nit-9A (V1M5) a, nit-9B (V1M32) A, nit-9C (V1M50) A NCU00498::hph+, mus52::bar+ a NCU00736::hph+, mus52::bar+ A NCU08375::hph+, mus52::bar+ A NCU03170::hph+, mus52::bar+ a NCU09746::hph+, mus52::bar+

nitrogen source was used to select for functionally complemented nit (nitrate non-utilizing) strains. For growth analysis on different reduced nitrogen sources, MM supplemented with nicotinate (15 mM) or hypoxanthine (2 mM) as sole N-source was used. For the molybdate reconstitution assay of nit-9 strains MM containing solely nitrate as N-source, supplemented with sodium molybdate (10 mM) was used. N. crassa strains were grown for 3–7 d at 30 °C. For solid MM 1.5% agar (w/v) was added. N. crassa liquid culture was performed in MM at 30 °C in the dark, with constant shaking at 130 rpm for 1 d. Crosses were performed on cornmeal medium (Westergaard and Mitchell, 1947) and incubated at RT until ejected ascospores were observed. Ascospores were grown on BDES plates at 30 °C overnight (Davis, 1970). Transformants were grown on FIGS medium at 30 °C (Chakraborty and Kapoor, 1990). 2.4. N. crassa creation of knock out strains Deletion cassettes for all Moco biosynthesis genes were constructed using a yeast recombinational cloning system (Colot et al., 2006). Primers used for the construction of the knock-out cassettes are shown in Supplementary Table 1. When applicable, primers suggested by the N. crassa knock-out consortium (Dunlap et al., 2007) were used. Knock-out cassettes were transformed into N. crassa mutants deficient in non-homologous end-joining DNA repair (mus-52::bar+ mat a) by electroporation. Subsequently, positive transformants were selected on hygromycin-containing FIGS medium. Transformants were picked onto VM agar slants containing hygromycin and then crossed to the N. crassa wildtype 74OR23-1A (mat A) strain on cornmeal medium. Ejected ascospores were harvested from each cross and plated on BDES agar plates containing sodium chlorate, thereby screening for an inactive NR. Grown ascospores were picked onto VM agar slants containing sodium chlorate and afterwards transferred to VM agar slants with phosphinothricin to identify progeny that harbored the mus-52 mutation (marked with bar, conferring resistance to phosphinothricin). Identification of the mating type of the generated Moco biosynthesis knock out strains was carried out as previously described (Perkins et al., 1989). Homokaryotic mutant strains were confirmed by Southern blot analysis and sequencing. Afterwards the homokaryotic mutant strains were analyzed for growth deficiency on MM without a reduced nitrogen source, thus documenting the lack of Moco dependent NR activity and on MM supplemented with hypoxanthine, thus documenting the lack of Moco dependent XDH activity. 2.5. Nucleic acid isolation and Southern hybridization Genomic DNA was isolated according to the method described by Lee and Taylor (1990). Southern hybridization was performed

72

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

as described (Sambrook and Russel, 2001). [32P] labeled probes were generated using random primed labeling (Hartmann Analytic, Braunschweig, Germany).

The extraction was performed at 4 °C and for 20 min. For clarifying, the crude extract was centrifuged for 20 min at 21,000g and 4 °C. The supernatant was transferred to a fresh reaction tube and used directly for further analysis.

2.6. Cloning of N. crassa Moco biosynthesis genes Cloning of the N. crassa Moco biosynthesis genes was carried out using locus specific primers (Supplementary Table 2) directed against the gene, including 500 bp of adjacent regulatory sequences. For amplification, the PhusionÒ High-Fidelity DNA Polymerase (NEB) was used. As PCR template, genomic DNA from N. crassa wild-type strain 74-OR23-1A (mat A) was used. Genomic DNA from this strain was extracted according to the method described by Lee and Taylor (1990). The CloneJET™ PCR Cloning Kit (Thermo Scientific) was used for subcloning according to the manufacturer’s instructions. 2.7. Immunoblot analysis For immunoblot analysis, protein extracts were prepared as described below und subjected to denaturing polyacrylamide gel electrophoresis using 12% (w/v) denaturating polyacrylamide gels. Upon blotting onto a PVDF membrane (GE healthcare), a Nit-9 primary antibody from mouse was used. An Anti-Mouse IgG + IgM secondary antibody conjugated to peroxidase (Dianova) was used to probe the Nit-9 specific antibody. 2.8. Total RNA extraction and cDNA synthesis Total RNA was extracted from grounded mycelia using the TRIzol Plus RNA Purification Kit (Ambion) or mirVana miRNA Isolation Kit (Ambion) according to manufacturer’s protocol followed by DNA-removal using TURBO DNA-free Kit (Ambion). For cDNA synthesis from total RNA M-MLV Reverse Transcriptase (Promega) or iScript cDNA Synthesis Kit (BioRad) were used according to manufacturer’s instructions. 2.9. Cloning of nit-7 cDNA For PCR based cloning of nit-7 cDNA the primers 50 - CGAGATCTGCTTCCTCATACCTGTCATCTG-30 and 50 -TCACTAGTCTCCCCATCCTGTCTCCTTG-30 were used. Specific amplification of the intron-containing nit-7 transcript was carried out using the primers 50 -GAGTTGGAGGGGTTCTGATATG-30 and 50 -CTAAAAGAACGCC CCAGTATGACC-30 . 2.10. Complementation of N. crassa nit strains For verification of annotated Moco biosynthesis mutant strains a functional complementation analysis was performed. The N. crassa Moco biosynthesis genes (i.e. the respective gene coding sequence and 500 bp of adjacent 50 and 30 regulatory sequences) were transformed into the different Moco biosynthesis nit mutant strains using electroporation, as previously described (Margolin et al., 1997). Positive transformants were screened using MM containing nitrate as sole source of nitrogen, thus selecting for Moco dependent NR activity. 2.11. Protein and metabolite extraction from N. crassa For protein and metabolite extraction, mycelium was grown in liquid culture for 1 d at 30 °C and 130 rpm. Afterwards the mycelium was harvested and ground in liquid nitrogen. Proteins and metabolites were extracted by adding ice cold extraction buffer (50 mM HEPES, 150 mM NaCl, 1%Triton (v/v), protease inhibitor (ROCHE Complete, EDTA-free), pH 7.2) and vigorously vortexing.

2.12. Proteomic analyses For the isolation of mitochondria, mycelium was grown and harvested as described above. The extraction protocoll is according Nargang and Rapaport (2007). In short the mycelium was ground at 4 °C in extraction buffer (250 mM sucrose, 25 mM HEPES, 5 mM EGTA (pH 7.5), protease inhhibitor (ROCHE Complete, EDTA-free) containing Silcondioxide (Sigma–Aldrich) and mitochondria were harvested by differential centrifugation at 4 °C in the presence of Benzonase. In the following a one-step 29% Percoll density gradient centrifugation takes place to enrich a pure mitochondira fraction Wieckowski et al. (2009). Integrity of mitochondria was checked by electron microscopy and cytochrome c oxidase assays (Cytochrom C Oxidase Assay Kit, Sigma–Aldrich). Proteins extracted from intact mitochondria were precipitated (Wessel and Flügge, 1984), redissolved in 8 M urea in 0.5 M TEAB-buffer and digested with a mixture of LysC and Trypsin (1 lg/ll). Peptides were desalted using RP18, labeled with iTRAQ™, separated by SCX and RP chromatography and sequenced on an OrbiTrap Velos (according to Scheiter et al. (2013)). Protein identification was supported by MASCOT (version 2.4) and manual MS data inspection by the Protein analysis platform of the HZI. The synthetic reference peptide was provide by the Peptide synthesis platform also at the HZI. 2.13. Moco metabolites detection and quantification Moco, and its metal-free precursors MPT and cPMP, were detected by conversion to their stable oxidation products FormA-dephospho (for Moco and MPT) and compound Z (for cPMP, Johnson and Rajagopalan, 1982; Johnson et al., 1989). Oxidation, dephosphorylation, QAE chromatography, and HPLC analysis were performed as described (Schwarz et al., 1997). First, a protein and metabolite extraction was performed as described above. For oxidation, 500 lg (total protein) of the clarified extract were used in a total volume of 400 ll 0.1 M Tris–HCl, pH 7.2. Oxidation was initiated upon addition of 50 ll acidic iodine (Schwarz et al., 1997). Following overnight oxidation, excess iodine was removed by the addition of 56 ll 1% ascorbic acid (w/v in water), and the sample was adjusted to pH 8.3 with 1 M Tris solution. The phosphate monoester of FormA was cleaved by calf intestine alkaline phosphatase (ROCHE) to obtain FormA-dephospho. FormA-dephospho and compound Z were further purified on QAE Sephadex A-25 columns. Subsequently, FormA-dephospho was eluted in a volume of 800 ll 10 mM acetic acid prior to elution of compound Z in 800 ll of 50 mM HCl. The resulting fractions were analyzed by injection onto a C-18 reversed-phase HPLC column. 3. Results 3.1. Identification of N. crassa Moco biosynthesis genes To reveal N. crassa gene loci involved in Moco biosynthesis, we first carried out a homology search using the primary sequences deduced from the A. nidulans Moco biosynthesis genes, which possesses a well annotated Moco biosynthesis pathway (Unkles et al., 1997, 1999; Appleyard et al., 1998; Millar et al., 2001; Heck et al., 2002). Using these A. nidulans sequences as queries in BLAST searches (Altschul et al., 1990) led to the identification of the respective N. crassa homologs (Table 2, for details please refer to

73

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

32

44 M al t-8 le le V1

ni

ni

al t-9B le le V1 M

7 54

A

al t-1 le le 34

1st 2nd

59

Moco biosynthesis step

nit-7 NCU03170 nit-8 nit-1 nit-9

ni

N. crassa

cnxABC cnxH cnxG cnxF cnxE

ni al t-7 le le V1

A. nidulans

synthase activity. Functional complementation of this strain was routinely possible using the cPMP synthase encoding gene (Fig. 2A, gene locus NCU00498). Data obtained from this functional

M

Table 2 Fungal Moco biosynthesis genes. The Aspergillus nidulans (A. nidulans) molybdenum cofactor (Moco) biosynthesis genes and their Neurospora crassa (N. crassa) homologs are listed.

3rd and 4th

control

the material and methods section). Next, functional domains were annotated, followed by a careful in silico analysis revealing catalytically important and thus strictly conserved residues known from Moco biosynthesis enzymes of various other species (Fig. 1). Once verified, we cloned the respective genes for functional complementation analysis of the known N. crassa Moco biosynthesis mutant strains.

NCU00498

NCU00736

The fungi N. crassa and A. nidulans, as well as humans, have one gene encoding for both first step proteins (hereafter referred to as cPMP synthase) necessary for cPMP synthesis, while plants (A. thaliana) have two. Consistently, there is only one N. crassa strain (harboring nit-7, allele V1M59) known which is impaired in cPMP Table 3 N. crassa Moco biosynthesis mutant strains. All known molybdenum cofactor (Moco) biosynthesis mutant strains are listed. For FGSC, strain numbers refer to the material and methods part. Locus

Allele

Linkage group

Study

nit-7 nit-1 nit-8 nit-9A nit-9B nit-9C nit-12

V1M59 34547 V1M44 V1M5 V1M32 V1M50 ko

IIIR IR IR IVR IVR IVR I

Tomsett and Garrett, Perkins (1959) Tomsett and Garrett, Tomsett and Garrett, Tomsett and Garrett, Tomsett and Garrett, This study

(1980) (1980) (1980) (1980) (1980)

32

ni

al t-9B le le V1 M

44

ni

al t-8 le le V1

M

7 54

ni

al t-1 le le 34

M ni al t-7 le le V1

NCU03170

54 7 al t-1 le le 34

ni

ni al t-9 le le ko

50 ni al t-9C le le V1 M

32

B

5

NCU09746

ni al t-9B le le V1 M

3.3. First step mutants

NCU08375

ni al t-9A le le V1 M

N. crassa Moco biosynthesis mutants are designated as nitrate non-utilizing (nit) mutants (Mendel and Schwarz, 2002). However, in addition to Moco deficiency, mutations in the nitrate assimilation pathway and its regulatory factors (Marzluf, 1997) can also cause a nit-phenotype. However, Moco is also a cofactor for several other Mo-enzymes (Schwarz et al., 2009), with xanthine dehydrogenase having – like NR – an easily selectable enzymatic activity. Therefore mutants in Moco biosynthesis display the pleiotropic loss of both enzymatic functions, thus making it rather easy to distinguish between these and mutations in the NR and XDH structural genes. Taking advantage of this, N. crassa Moco biosynthesis mutant strains have been identified followed by mapping of the mutated gene loci (Tomsett and Garrett, 1980; Perkins et al., 2001, Table 3). Upon availability of the N. crassa genome sequence, and completion of its annotation (Galagan et al., 2003; Dunlap et al., 2007), the gene loci predicted to encode Moco biosynthesis enzymes have been assigned to the Moco biosynthesis mutant strains using the genetic mapping data available (McCluskey et al., 2010; Dunlap et al., 2007). The following set of experiments aims to functionally complement N. crassa nit mutants. Therefore, the before identified Moco biosynthesis gene loci have been cloned (including ±500 bp upand downstream of start and stop codon respectively) and sequenced, thus verifying their sequence integrity.

59

3.2. Functional complementation of N. crassa Moco biosynthesis mutants

-

+

Fig. 2. Functional complementation of N. crassa Moco biosynthesis strains. (A) Neurospora crassa (N. crassa) nit (nitrate non-utilizing) mutant strains were each transformed with the molybdenum cofactor (Moco) biosynthesis encoding genes. As a control, the transformation was performed with water instead of DNA. Functional complementation was monitored by the ability of the transformants to use nitrate as nitrogen source, thus documenting Moco-dependent NR activity. (B) Molybdate dependent growth of of nit-9 mutant strains. For molybdate dependent reconstitution, molybdate (to a concentration of 10 mM) was added (+) to Vogel’s medium containing solely nitrate as N-source. As negative control, the Moco biosynthesis mutant strain harboring nit-1 allele 34547 was used.

74

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

complementation experiment confirmed available biochemical (Heck and Ninnemann, 1995), mapping, and annotation (Tomsett and Garrett, 1980; Perkins et al., 2001) data. 3.4. Second step mutants The second step of Moco biosynthesis requires the complex interplay of three different proteins, necessary for the conversion of cPMP to MPT. We have identified the gene loci NCU03170 and NCU08375 as encoding the large and small subunit of the MPT synthase, respectively, and NCU00736 was found to code for the MPT synthase sulfurase. However, to our knowledge, only two strains (harboring nit-8 allele V1M44 and nit-1 allele 34547 respectively) which have the biochemical properties of a second step mutant have been previously described (Dunn-Coleman, 1984; Heck and Ninnemann, 1995). Functional complementation reliably identified the nit-8 allele V1M44 harboring strain as being mutated in the gene coding for the MPT-synthase small subunit (NCU08375, Fig. 2A), but contrary to our results, this allele was previously suggested to encode a mutated variant of the MPT-synthase large subunit (encoded by the gene locus NCU03170, Perkins et al., 2001). The other available second step mutant strain (harboring nit-1 allele 34547) has been functionally complemented by the gene coding for the MPT-synthase sulfurase (gene locus NCU00736, Fig. 2A), confirming previous biochemical (Dunn-Coleman, 1984; Heck and Ninnemann, 1995), mapping and annotation (Perkins, 1959; Tomsett and Garrett, 1980; Perkins et al., 2001) data. 3.5. Third and fourth step mutants The third and fourth steps of Moco biosynthesis are intrinsically connected. In eukaryotes, both steps are catalyzed by one enzyme which possesses two functional domains (called E- and G-domain). The Moco intermediate MPT-AMP was identified only recently, resulting in the re-annotation of Moco biosynthesis as a four-step pathway (Llamas et al., 2006; Schwarz et al., 2009). In most of the currently available literature however, Moco biosynthesis is characterized as three-step pathway, and consequently any mutation related to the Mo insertion into the MPT backbone has been designated as a third-step mutation. For clarity, from this point on we will refer to the original annotation of Moco biosynthesis as three-step pathway. There are three known third-step Moco mutant strains (harboring nit-9A allele V1M5, nit9-B, allele V1M32 or nit9-C allele V1M50 respectively), which have been identified based on biochemical characterization (Dunn-Coleman, 1984; Heck and Ninnemann, 1995). Results from our functional complementation experiments only partly confirmed the biochemical data sets obtained for these strains, since upon transformation with the gene encoding for the Mo-insertase (gene locus NCU09746), only the strain harboring nit9B allele V1M32 routinely showed the ability to be complemented (Fig. 2A). Results from functional complementation experiments with strains harboring nit-9A allele V1M5 or nit-9C allele V1M50 were inconsistent and not reproducible. Mapping data obtained for strains harboring nit-9-A, allele V1M5 or nit-9B, allele V1M32 assigned the underlying mutation(s) to the same locus (NCU09746, Perkins et al., 2001; Tomsett and Garrett, 1980). Previous positive selection of heterokaryon growth suggests that nit-9B, allele V1M32 may also map to this locus, however further data is needed for confirmation (Tomsett and Garrett, 1980). Nevertheless, results from our functional complementation assay confirm the annotation of NCU09746 as Mo-insertase, although the position of the gene locus NUC09746 in the strain harboring nit-9B, allele V1M32 may be in dispute.

To further validate the results obtained from our functional complementation experiments, we next tested the three strains harboring the nit-9 alleles V1M5, V1M32 or V1M50 and a strain carrying a nit-9 knock out allele created in this study for molybdate dependent growth on nitrate selective media (Arst and Cove, 1970). Therefore, an excess of molybdate was added to media containing solely nitrate as N-source and used for cultivation of the strains harboring the different nit-9 alleles. Upon prolonged incubation, growth was detectable, thus documenting the Moco dependent NR activity (Fig. 2B). High cellular molybdate concentrations result in the Moco synthase independent conversion of MPT to Moco. Consistently, no growth was detectable for the strain harboring the nit-1 allele 34547 used as negative control in this experiment. 3.6. Sequencing of the N. crassa Moco biosynthesis mutants In order to further characterize the mutations harbored in the various Moco biosynthesis nit mutants, we cloned and sequenced the mutated genes. PCR-based cloning was performed from the predicted start to the predicted stop codon of each gene. Results from the sequencing and the predicted effects of the identified mutations at the protein level are summarized in Table 4. The position of the identified mutations in the genes is illustrated in Fig. 1. 3.7. Creation of Moco biosynthesis knock out strains Genetic characterization of the N. crassa Moco biosynthesis nit mutants experimentally assigned the mutated Moco biosynthesis genes to the various nit mutant strains. In doing so we revealed that, for the gene encoding the MPT synthase large subunit, a corresponding nit mutant strain is missing. However, our homology search identified locus NCU03170 to code for the MPT synthase large subunit. For experimental validation, we created and characterized a corresponding mutant. The gene locus NCU03170 was knocked out, using the method described by Colot et al. (2006). For comparison, we also knocked out the remaining N. crassa Moco biosynthesis genes, yielding a complete set of genetically fully defined N. crassa Moco biosynthesis knock-out strains. Primary heterokaryotic transformants were backcrossed and selected for chlorate tolerance growth. To genetically characterize the homokaryotic knock out strains, we first analyzed them using Southern blotting, verifying the single integration of the knock-out cassette at the correct locus. Subsequently, we confirmed the integrity of

Table 4 Mutations identified in the N. crassa nit-mutant alleles. Mutations of all Neurospora crassa (N. crassa) nit- (nitrate non-utilizing) mutant alleles and the predicted effects of the identified mutations at the protein level are summarized.a For nit-1 allele 34547 only exon located mutations are listed in this table, in Supplementary Table 3 also the intron located mutations are summarized.b nit-9B V1M32 is not expressed as its gene product has not been detected by western blotting (Supplementary Fig. 3). Allele

Mutation

Effect at protein level

nit-7 V1M59

A411G, DT413

nit-8 V1M44

DT249

Premature termination of translation at aa 163 Elongation of the protein by 29 aa D28E, V82A, D253T, D254T, T257A, V266M, D375N, R425L

nit-1 34547

a

C84A, C105T, C352T, T629C, C662T, A746G, A870G, D878883 (CTACTA), G903A, T1100C, G1230A, G1381T, C1382T A1285T

nit-9A V1M5 nit-9B V1M32 nit-9C V1M50

b

– DG113, DC114

Premature termination of translation at aa 482 – Premature termination of the protein at aa 37

75

ko 74

media containing

wt

NC U0 9

0 17 NC U0 3

6

ko

ko 5 37 NC U0 8

73

8

NC U0 0

49 NC U0 0

A

6

ko

ko

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

NH4+ + NO3-

NO 3-

NH4+ + NO3- ClO3-

hypoxanthine

1 s t s te p

B

2 nd step

3 r d s te p

67.5

pmol pterin per mg crude extract

60 52.5 45 37.5 30 22.5 15

cPMP Moco/MPT

7.5 0

ND ND

ND

ND

ND

NCU00498 NCU00736 NCU08375 NCU03170 NCU09746

wt

ko strains Fig. 3. Moco biosynthesis knock out strains. (A) Growth phenotype of molybdenum cofactor (Moco) biosynthesis knock-out (ko) strains on media containing different N-sources. (B) Detectable Moco/MPT (molybdopterin) and cyclic pyranopterin monophosphate (cPMP) in crude extracts from the Neurospora crassa (N. crassa) knock-out strains shown in (A).

neighboring genes by sequencing the knock-out locus and its adjacent genes (data not shown). 3.8. Biochemical characterization of Moco biosynthesis knock out strains Following genetic validation, we characterized the growth phenotype of the knock-out strains (Fig. 3A). As expected, no growth was detectable when cultivated on media containing solely nitrate as N-source. Consistently, all Moco biosynthesis knock-out strains were able to grow on chlorate-containing media, confirming their loss of NR activity. To validate that our Moco biosynthesis knock out strains display the characteristic pleiotropic loss of both, NR and XDH activity (Mendel, 2013), we next documented their growth on media containing hypoxanthine as sole N-source. Consistent with published data, the tested Moco biosynthesis knock out strains did not grow on media containing solely hypoxanthine as N-source, thus exhibiting the expected growth phenotype. Next to NR and XDH amongst fungi at least A. nidulans houses the Moco dependent enzyme nicotinate hydroxylase (Scazzocchio et al., 1973), having like NR and XDH an easily detectable enzymatic activity. In order to test for this enzymatic activity, the N. crassa wildtype strain, as well as the created Moco biosynthesis knock out mutants was grown on selective media containing nicotinate

as sole N-source. However, neither for the wildtype, nor for the Moco mutant strains growth was detectable (data not shown), thus documenting that other than A. nidulans, N. crassa houses no nicotinate hydroxylase. In the following, we quantified the detectable Moco metabolites cPMP and Moco/MPT (Fig. 3B). As expected, neither cPMP nor MPT/ Moco were detectable in the cPMP synthase deficient NCU00498 knock-out strain. The NCU03170 knock-out strain possesses no Moco/MPT but accumulates cPMP, similar to the biochemical properties of the second step knock-out strains NCU00736 ko (MPT synthase sulfurase) and NCU08375 ko (MPT synthase large subunit). Therefore we conclude that the NCU03170 locus does in fact encode for the MPT synthase large subunit, verifying our annotation model. 4. Discussion In the past, eukaryotic Mo metabolism has been studied intensively, using primarily the higher plants A. thaliana and Nicotiana plumbaginifolia as model systems (Poll et al., 1991; Mendel, 1997; Mendel and Hänsch, 2002; Mendel and Kruse, 2012). Defects in plant Mo metabolism are accompanied by severe phenotypes that frequently complicate plant cultivation and sexual reproduction. Indeed, mutant plants were found to be valuable tools for

76

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

elucidating eukaryotic Moco biosynthesis, since only the combined characterization of mutants and of the enzymes involved in Moco biosynthesis led to the present in-depth understanding of this evolutionarily old and highly-conserved pathway (Stallmeyer et al., 1995, 1999; Schwarz et al., 2000). Although mechanistically characterized in detail (Mendel and Kruse, 2012), general questions addressing the principles of allocation and transfer of Moco and its insertion into target enzymes are still unanswered (Fischer et al., 2006; Kruse et al., 2010). New strategies are therefore needed to reveal yet unidentified players and interaction partners involved in eukaryotic Mo metabolism. This requires a genetically easy to manipulate model system which can also tolerate the loss of Moco biosynthesis. The filamentous fungus N. crassa fulfills both requirements. However, using N. crassa to address these questions essentially depends on a reliably annotated Moco biosynthesis pathway. We identified the N. crassa Moco biosynthesis pathway as encoded by five genes, agreeing with the number of Moco biosynthesis genes identified in the fungus A. nidulans (Unkles et al., 1997, 1999; Appleyard et al., 1998; Millar et al., 2001; Heck et al., 2002). However, human Moco biosynthesis is encoded by four genes and plant Moco biosynthesis by six (Mendel and Kruse, 2012). What are the reasons for these discrepancies? Plants encode the two first-step enzymes Cnx2 and Cnx3 by separate genes, while the homologous proteins from humans were shown to be encoded by the bicistronic mocs1 transcript. Similar to humans, both N. crassa and A. nidulans have only one gene assigned to the first step of Moco biosynthesis, thus raising the question how this single gene can encode for the two first step enzymes of Moco biosynthesis. The A. nidulans first step gene cnxABC was suggested to encode a fusion protein containing both catalytic domains required for cPMP synthesis (Unkles et al., 1997). However, work with the human first step enzymes revealed the Cnx2 homologous protein Mocs1A to depend on a C-terminal double glycine motif for catalytic activity and consistently, missense mutations in this motif were found to be causal for Moco deficiency (Hänzelmann and Schindelin, 2004). Consistent with these findings, Hänzelmann et al. (2002) identified a human fusion protein comprising both catalytic domains but lacking the functionally important double glycine motif and therefore being catalytically inactive (Hänzelmann et al., 2002). Both, N. crassa and A. nidulans first step enzymes lack the

double glycine motif when expressed as a fusion protein as suggested by Unkles et al. (1997). Did fungi develop a different mechanism for GTP conversion to cPMP or is there another explanation? Careful analysis of the N. crassa nit-7 gene sequence identified the catalytically important double glycine motif to be intron-coded. Here, the extension of exon 1 into the adjacent intron 1 by 8 nucleotides would result in the formation of the double glycine motif containing N. crassa counterpart of Mocs1A/Cnx2 (Fig. 4). The same has been suggested for the Mocs1A/Cnx2 homolog from A. nidulans (CnxABC, Reiss, 2000). Consistently for the first step genes of N. crassa and A. nidulans an intron-located termination codon has been identified following the codons encoding for the double glycine motif. Consequently, we assume the N. crassa Mocs1A counterpart to be expressed from an intron-containing splice variant. To prove our assumption we first sequenced the nit-7 cDNA, revealing exclusively the intron-free (spliced) variant of the mRNA. Consistently also its amplification with intron-specific primer pairs failed. We therefore conclude that the assumed intron- containing messenger is (i) either only present in marginal amounts or (ii) readily degraded upon formation. Consequently in a following experiment we employed mass spectrometry to prove the existence of the double glycine-containing N. crassa Mocs1A/Cnx2 homolog. The first step of eukaryotic Moco biosynthesis is located in the mitochondria, thus LC–MS based experiments were carried out using isolated mitochondria from N. crassa. Hereby peptides mapping to both Nit-7 catalytic domains were identified (data not shown). Amongst these, a C-terminal double glycine motif containing peptide was identified upon comparison with the fingerprint of the respective synthetic peptide (Supplementary Fig. 1), thus unambiguously confirming the presence of the postulated (double glycine motif containing) N. crassa Mocs1A/Cnx2 homolog. How can the presence of the fusion-protein encoding messenger of N. crassa and A. nidulans be explained? The human Cnx3 homolog (MOCS1B) was identified to be expressed from mocs1 splice forms II and III, yielding a MOCS1AB fusion protein with an inactive A domain and functional B domain (Reiss and Hahnewald, 2011). Translating this finding to N. crassa and A. nidulans suggests that the two first step enzymes are also expressed as different splice variants of one messenger, resulting (i) in the synthesis of a fusion protein containing an inactive Mocs1A/Cnx2 homolog but active Mocs1B/Cnx3 domain (intron-free messenger)

A

B

Fig. 4. The N. crassa cPMP synthase double glycine motif. (A) Detail of the Neurospora crassa (N. crassa) NCU00498 and Aspergillus nidulans (A. nidulans) cnxABC gene loci. Exonic regions are shaded in dark gray, and the sequence is written with white letters; intronic regions are shaded in light gray with the sequence written in black letters. The encoded amino acids are given below the coding sequence; two asterisks mark the catalytically important C-terminal double glycine motif. (B) Sequence comparison of the C-terminal 12 amino acid residues from Nit-7 (N. crassa, NCU00498), CnxABC (A. nidulans, NCBI Reference Sequence: XP_658551.1), Mocs1A (Homo sapiens, GenBank entry AAI40422.1), Cnx2 (Arabidopsis thaliana, GenBank entry AAM64798.1) and MoaA Escherichia coli (GenBank entry CAA49861.1).

77

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

N. crassa

N

G-domain

E-domain

C

A. nidulans

N

G-domain

E-domain

C

H. sapiens

N

A. thaliana

N

E. coli

N

E-domain

G-domain

E-domain

E-domain

C

G-domain

C

N

C

G-domain

C

Fig. 5. Domain organization of eukaryotic and prokaryotic Mo-insertases. The domain organization of the Mo-insertases from Neurospora crassa (N. crassa), Aspergillus nidulans (A. nidulans), Homo sapiens (H. sapiens) and Arabidopsis thaliana (A. thaliana) is shown. The functionally important Mo-insertase domains (E and G) are organized in one protein in eukaryotes, while in prokaryotes as Escherichia coli (E. coli) they are expressed as separate proteins.

Table 5 Annotation of the N. crassa Moco = molybdenum cofactor.

5. Conclusions Moco

biosynthesis.

nit = nitrate

non-utilizing;

nit-Nomenclature

Gene locus

Coding for

Moco biosynthesis step

nit-7 nit-1 nit-8 nit-12 nit-9

NCU00498 NCU00736 NCU08375 NCU03170 NCU09746

Precursor Z synthase MPT synthase sulfurase MPT synthase small subunit MPT synthase large subunit Mo-insertase

1st 2nd 2nd 2nd 3rd and 4th

and (ii) in the formation of the double glycine motif containing Mocs1A/Cnx2 homolog (intron-containing messenger). In addition to the first step encoding human mocs1 gene, the second step encoding human mocs2 gene is also known to encode a bicistronic messenger (Reiss and Hahnewald, 2011). Mocs2 encodes the large and small subunit of MPT-synthase. However, in plants (Arabidopsis and Nicotiana) as well as in the fungus A. nidulans, previous work identified each subunit as encoded by a separate gene. In our work, we identified both subunits of the N. crassa MPT synthase as encoded by single genes as well, in agreement with the gene organization pattern identified previously in A. nidulans and plants. The third step of eukaryotic Moco biosynthesis is catalyzed by two functional domains of one enzyme, encoded by a single gene in plants (with the exception of the green alga Chlamydomonas reinhardtii, Llamas et al., 2007), fungi and humans. Differences exist concerning the functional domain organization (Fig. 5): While the Moco synthase from plants consists of an N-terminal E- and C-terminal G-domain, the homolog from humans shows an inverted domain organization. For N. crassa and A. nidulans the domain organization was found to be similar to that of humans. In general, the gene organization of N. crassa and A. nidulans Moco biosynthesis appears to be more similar to that of humans than to that of plants. This can be explained by the closer phylogenetic relationship between fungi and animals, which are believed to share a common unicellular ancestor (Wainright et al., 1993). However, addressing this question is clearly beyond the scope of this work, but has potential to shed light on the evolutionary reasons for differences in gene organization patterns identified for Moco biosynthesis in eukaryotes.

Moco biosynthesis is a multi-step pathway consisting of four reaction steps. Six enzymes are necessary for the synthesis of Moco in eukaryotes, whereas the number of encoding genes varies amongst plants, fungi and humans (Supplementary Fig. 2). In this work, we completed and corrected the previous annotation of N. crassa Moco biosynthesis. We suggest retaining the N. crassa nit nomenclature for Moco biosynthesis genes. Notably, to our knowledge no nit mutant strain defective in the MPT synthase large subunit has been described, yet the gene has already been named nit-12 (McCluskey et al., 2010). The complete annotation of N. crassa Moco biosynthesis is summarized in Table 5. Acknowledgments We thank André Fleißner (from the Department of Genetics, Braunschweig University of Technology) for carefully reading the manuscript and Ewald Priegnitz (from the Department of Genetics, Braunschweig University of Technology) for his support with Southern blotting. We thank Anke Oelbermann (from the Department of Plant Biology, Braunschweig University of Technology) for excellent technical assistance. We thank Sabine Buchmeier (from the Institute of Physical and Theoretical Chemistry, Braunschweig University of Technology) for the excellent Nit-9 antibody. We also thank the Protein analytics platform at the HZI for support with the MS analyses and the peptide synthesis platform for supplying the Nit-7 reference peptide. We acknowledge the FGSC (Kansas City, Missouri USA) for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2014.02.004. References Altschul, S.F. et al., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403– 410. Appleyard, M.V. et al., 1998. The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis. Molecular characterization and analysis of in vivo generated mutants. J. Biol. Chem. 273, 14869–14876.

78

C. Probst et al. / Fungal Genetics and Biology 66 (2014) 69–78

Arst Jr., H.N., Cove, D.J., 1970. Molybdate metabolism in Aspergillus nidulans. II. Mutations affecting phosphatase activity or galactose utilization. Mol. Gen. Genet. 108, 146–153. Borkovich, K.A. et al., 2004. Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol. Mol. Biol. Rev. 68, 1–108. Chakraborty, B.N., Kapoor, M., 1990. Transformation of filamentous fungi by electroporation. Nucleic Acids Res. 18, 6737. Coddington, A., 1976. Biochemical studies on the nit mutants of Neurospora crassa. Mol. Gen. Genet. 145, 195–206. Colot, H.V. et al., 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA 103, 10352–10357. Cove, D.J., Pateman, J.A., 1963. Independently segregating genetic loci concerned with nitrate reductase activity in Aspergillus nidulans. Nature 198, 262–263. Davis, R.H.D.S.F.J., 1970. Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol. 17A, 79–143. Dunlap, J.C. et al., 2007. Enabling a community to dissect an organism: overview of the Neurospora functional genomics project. Adv. Genet. 57, 49–96. Dunn-Coleman, N.S., 1984. Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity. Curr. Genet. 8, 581–588. Fischer, K. et al., 2006. Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii. J. Biol. Chem. 281, 30186–30194. Fleißner, A., 2012. Hyphal Fusion. Topics in Current Genetics, vol. 22. SpringerVerlag, pp. 43–60. Galagan, J.E. et al., 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868. Hänzelmann, P. et al., 2002. Functionality of alternative splice forms of the first enzymes involved in human molybdenum cofactor biosynthesis. J. Biol. Chem. 277, 18303–18312. Hänzelmann, P., Schindelin, H., 2004. Crystal structure of the Sadenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans. Proc. Natl. Acad. Sci. USA 101, 12870–12875. Heck, I.S., Ninnemann, H., 1995. Molybdenum cofactor biosynthesis in Neurospora crassa: biochemical characterization of pleiotropic molybdoenzyme mutants nit-7, nit-8, nit-9A, B and C. Photochem. Photobiol. 61, 54–60. Heck, I.S. et al., 2002. Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans. Genetics 161, 623–632. Hu, Y., Ribbe, M.W., 2011. Biosynthesis of Nitrogenase FeMoco. Coord. Chem. Rev. 255, 1218–1224. Johnson, J.L., Rajagopalan, K.V., 1982. Structural and metabolic relationship between the molybdenum cofactor and urothione. Proc. Natl. Acad. Sci. USA 79, 6856– 6860. Johnson, J.L. et al., 1989. The structure of a molybdopterin precursor. Characterization of a stable, oxidized derivative. J. Biol. Chem. 264, 13440– 13447. Kaufholdt, D. et al., 2013. Visualization and quantification of protein interactions in the biosynthetic pathway of molybdenum cofactor in Arabidopsis thaliana. J. Exp. Bot. 64, 2005–2016. Kruse, T. et al., 2010. Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana. J. Biol. Chem. 285, 6623–6635. Kuper, J. et al., 2004. Structure of molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism. Nature 430, 806. Lee, SB., Taylor, JT., 1990. Isolation of DNA from fungal mycelia and single spores. In: Innis, M.A., Gelfand, DH., Sninsky, JJ., White, TJ. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, NY, pp. 82–287. Llamas, A. et al., 2004. Synthesis of adenylated molybdopterin: an essential step for molybdenum insertion. J. Biol. Chem. 279, 55241–55246. Llamas, A. et al., 2006. The mechanism of nucleotide-assisted molybdenum insertion into molybdopterin: novel routes towards metal cofactor assembly. J. Biol. Chem. 281, 18343-18340. Llamas, A. et al., 2007. Chlamydomonas reinhardtii CNX1E reconstitutes molybdenum cofactor biosynthesis in Escherichia coli mutants. Eukaryot. Cell 6, 1063–1067. Margolin, B.S., Freitag, M., Selker, E.U., 1997. Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Gen. Newslett. 44, 34–36. Marzluf, G.A., 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiol. Mol. Biol. Rev. 61, 17–32. McCluskey, K. et al., 2010. The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J. Biosci. 35, 119–126. Mendel, R.R., 1997. Molybdenum cofactor of higher plants: biosynthesis and molecular biology. Planta 203, 399–405. Mendel, RR., 2013. The molybdenum cofactor. J. Biol. Chem. Mendel, R.R., Hänsch, R., 2002. Molybdoenzymes and molybdenum cofactor in plants. J. Exp. Bot. 53, 1689–1698. Mendel, R.R., Kruse, T., 2012. Cell biology of molybdenum in plants and humans. Biochim. Biophys. Acta 1823, 1568–1579. Mendel, R.R., Schwarz, G., 2002. Biosynthesis and molecular biology of the molybdenum cofactor (Moco). Met. Ions Biol. Syst. 39, 317–368.

Millar, L.J. et al., 2001. Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans. Mol. Genet. Genomics 266, 445–453. Nargang, F.E., Rapaport, D., 2007. Neurospora crassa as a model organism for mitochondrial biogenesis. Methods Mol. Biol. 372, 107–123. Pateman, J.A. et al., 1964. A common cofactor for nitrate reductase and xanthine dehydrogenase wich also regulates the synthesis of nitrate reductase. Nature 201, 58–60. Perkins, D.D., 1959. New markers and multiple point linkage data in Neurospora. Genetics 44, 1185–1208. Perkins, D.D. et al., 1989. Neurospora strains incorporating fluffy, and their use as testers. Fungal Genet. Newslett. 36, 64–66. Perkins, D.D. et al., 2001. The Neurospora Compendium: Chromosomal Loci. Academic Press Inc., San Diego, Calif. Poll, A.M. et al., 1991. Biochemical characterization of cnx nitrate reductasedeficient mutants from Nicotiana plumbaginifolia. Plant Sci. 76, 201–209. Rajagopalan, K.V., Johnson, J.L., 1992. The pterin molybdenum cofactors. J. Biol. Chem. 267, 10199–10202. Reiss, J., 2000. Genetics of molybdenum cofactor deficiency. Hum. Genet. 106, 157– 163. Reiss, J., Hahnewald, R., 2011. Molybdenum cofactor deficiency: mutations in GPHN, MOCS1, and MOCS2. Hum. Mutat. 32, 10–18. Rieder, C. et al., 1998. Rearrangement reactions in the biosynthesis of molybdopterin-an NMR study with multiply 13C/15N labelled precursors. Eur. J. Biochem. 255, 24–36. Ringel, P. et al., 2013. Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa. J. Biol. Chem. 288, 14657–14671. Sambrook, J., Russel, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York. Scazzocchio, C. et al., 1973. The genetic control of molybdoflavoproteins in Aspergillus nidulans. Allopurinol-resistant mutants constitutive for xanthinedehydrogenase. Eur. J. Biochem. 36, 428–445. Scheiter, M. et al., 2013. Proteome analysis of distinct developmental stages of human natural killer (NK) cells. Mol. Cell. Proteomics 12, 1099–1114. Schwarz, G. et al., 1997. Molybdenum cofactor biosynthesis. The plant protein Cnx1 binds molybdopterin with high affinity. J. Biol. Chem. 272, 26811–26814. Schwarz, G. et al., 2009. Molybdenum cofactors, enzymes and pathways. Nature 460, 839–847. Schwarz, G. et al., 2004. Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli. Hum. Mol. Gen. 13, 1249–1255. Schwarz, G. et al., 2000. The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton. Plant Cell 12, 2455–2472. Seefeldt, L.C. et al., 2009. Mechanism of Mo-dependent nitrogenase. Annu. Rev. Biochem. 78, 701–722. Seiler, S., Plamann, M., 2003. The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa. Mol. Biol. Cell 14, 4352–4364. Seiler, S. et al., 1999. Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora. Curr. Biol. 9, 779–785. Stallmeyer, B. et al., 1995. Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins. Plant J. 8, 751–762. Stallmeyer, B. et al., 1999. The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells. Proc. Natl. Acad. Sci. USA 96, 1333–1338. Teschner, J. et al., 2010. A novel role for Arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis. Plant Cell 22, 468–480. Tomsett, A.B., Garrett, R.H., 1980. The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa. Genetics 95, 649–660. Unkles, S.E. et al., 1997. The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis. J. Biol. Chem. 272, 28381– 28390. Unkles, S.E. et al., 1999. Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants. J. Biol. Chem. 274, 19286–19293. Veldman, A. et al., 2010. Successful treatment of molybdenum cofactor deficiency type A with cPMP. Pediatrics 125, e1249–e1254. Vogel, H., 1956. A convenient growth medium for Neurospora (Medium N). Microb. Genet. Bull. 13, 42–43. Wainright, P.O. et al., 1993. Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260, 340–342. Wessel, D., Flügge, U.I., 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143. Westergaard, M., Mitchell, H.K., 1947. Neurospora V. A synthetic medium favoring sexual reproduction. Am. J. Bot. 34, 573–577. Wieckowski, M.R. et al., 2009. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4, 1582–1590. Wuebbens, M.M., Rajagopalan, K.V., 1993. Structural characterization of a molybdopterin precursor. J. Biol. Chem. 268, 13493–13498.