Ulrich Schulte

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

SUMMARY We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Biogenesis of respiratory complex I.

Proteins specifically involved in the biogenesis of respiratory complex I in eukaryotes have been characterized. The complex I intermediate associated proteins CIA30 and CIA84 are tightly bound to an assembly intermediate of the membrane arm. Like chaperones, they are involved in multiple rounds of membrane arm assembly without being part of the mature structure. Two biosynthetic subunits of eukaryotic complex I have been characterized. The acyl carrier subunit is needed for proper assembly of the peripheral arm as well as the membrane arm of complex I. It may interact with enzymes of a mitochondrial fatty acid synthetase. The 39/40-kDa subunit appears to be an isomerase with a tightly bound NADPH. It is related to a protein family of reductases/isomerases. Both subunits have been discussed to be involved in the synthesis of a postulated, novel, high-potential redox group.

Redox components and structure of the respiratory NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase is the first complex in the respiratory chains of many purple bacteria and of mitochondria of most eucaryotes. The bacterial complex consists of 14 different subunits. The mitochondrial complex contains at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. We analysed electron micrographs of isolated and negatively stained complex I particles from Escherichia coli and Neurospora crassa and obtained three-dimensional models of both complexes at medium resolution. Both have the same L-shaped overall structure with a peripheral arm protruding into the aqueous phase and a membrane arm extending into the membrane. The two arms of the bacterial complex are only slightly shorter than those of the mitochondrial complex although the protein mass of the former is only half of that of the latter. The presence of a novel redox group in the membrane arm of the complex is discussed. This group has been detected in the N. crassa complex by means of UV-visible spectroscopy. After reduction with an excess of NADH and reoxidation by the lactate dehydrogenase reaction, a reduced-minus-oxidized difference spectrum was obtained that cannot be attributed to the known cofactors flavin mononucleotide (FMN) and the FeS clusters N1, N2, N3 and N4. Due to its positive midpoint potential the novel group is believed to transfer electrons from the FeS clusters to ubiquinone. Its role in proton translocation is discussed.

[1] Generation and characterization of NADH: Ubiquinone oxidoreductase mutants in Neurospora crassa

Publisher Summary The electron microscopy of the N. crassa complex I showed an unusual L-shaped structure with the shorter arm of the L protruding into the matrix and the larger embedded in the mitochondrial inner membrane. The bipartite structure is reflected in the functional organization of the complex, in the organization of the genes, and in the mechanism of assembly. The exclusively nuclear-encoded peripheral arm, including the Flavin mononucleotide (FMN) and four Iron–sulfur protein (FeS) clusters contains the Nicotinamide adenine dinucleotide proton (NADH) dehydrogenase part, whereas the membrane arm with all seven mitochondrially encoded subunits constitutes the ubiquinone hydrogenase part. Assembly of the complex involves the formation of independent intermediates, which join just as building blocks in the stepwise formation of the complex. This chapter discusses the generation of complex I mutants by gene disruption and the immunoprecipitation of incompletely assembled parts of complex I.

Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.

Disruption of iron-sulphur cluster N2 from NADH:ubiquinone oxidoreductase by site-directed mutagenesis

We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3 kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group.

The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria.

An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination.

Large scale analysis of sequences from Neurospora crassa

After 50 years of analysing Neurospora crassa genes one by one large scale sequence analysis has increased the number of accessible genes tremendously in the last few years. Being the only filamentous fungus for which a comprehensive genomic sequence database is publicly accessible N. crassa serves as the model for this important group of microorganisms. The MIPS N. crassa database currently holds more than 16 Mb of non-redundant data of the chromosomes II and V analysed by the German Neurospora Genome Project. This represents more than one-third of the genome. Open reading frames (ORFs) have been extracted from the sequence and the deduced proteins have been annotated extensively. They are classified according to matches in sequence databases and attributed to functional categories according to their relatives. While 41% of analysed proteins are related to known proteins, 30% are hypothetical proteins with no match to a database entry. The entire genome is expected to comprise some 13000 protein coding genes, more than twice as many as found in yeasts, and reflects the high potential of filamentous fungi to cope with various environmental conditions.

Identification of the TYKY homologous subunit of complex I from Neurospora crassa.

A polypeptide subunit of complex I from Neurospora crassa, homologous to bovine TYKY, was expressed in Escherichia coli, purified and used for the production of rabbit antiserum. The mature mitochondrial protein displays a molecular mass of 21280 Da and results from cleavage of a presequence consisting of the first 34 N-terminal amino acids of the precursor. This protein was found closely associated with the peripheral arm of complex I.

Two pentatricopeptide repeat domain proteins are required for the synthesis of respiratory complex I

In this study pentatricopeptide repeat (PPR) proteins in filamentous ascomycetes are identified and functionally characterized. PPR proteins, which have in common a degenerated 35 amino acid motif often arranged in multiple tandems, are known to be implicated in various steps of RNA metabolism in mitochondria and chloroplasts. In filamentous ascomycetes we identified a common set of nine PPR proteins. For seven of these proteins, which were not yet characterized, knockout mutants of Neurospora crassa were analyzed. The knockout of three genes appeared to be lethal while four mutants showed different degrees of alterations in respiratory chain complexes. Two mutants are specifically affected in the assembly of a functional complex I while the other enzymes of the respiratory chain are present. Both mutants demonstrate the presence of a peripheral arm and the absence of a detectable membrane arm. Analysis of the mitochondrial RNA revealed distinct alterations of the transcript patterns for certain complex I subunits. Synthesis and/or stability of the transcript for ND2–ND3 is grossly impaired in one mutant while in the other mutant splicing of the transcript for ND1–ND4 is hampered. Our analysis provides the basis for a comprehensive characterization of PPR proteins in filamentous ascomycetes.