Plenary session abstracts


Vegetative incompatibility in Podospora anserina, characterization of genes induced during non-allelic incompatibility. Joël Bégueret, Institut CNRS de Biochimie et Génétique Cellulaires, Bordeaux, France.

In P. anserina, nine het loci have been identified by genetic analysis of wild-type isolates. Five of these loci are involved in allelic incompatibility systems as described in Neurospora. crassa and other fungi. However in Podospora, genetic interactions between non-allelic het genes have also been described. In these cases, the incompatibility reaction is triggered by the coexpression of het genes that belong to different loci. Five het genes involved in such non-allelic incompatibility systems have been identified, they define three incompatibility systems, het-R/het-V, het-C/het-E and het-C/het-D. We have used the thermosensitivity of the het-R/het-V system to identified genes whose expression is induced during the progress of the incompatibility reaction. Four genes, named idi (for induced during incompatibility) have been characterized. idi-1, 2 and 3 encode small polypeptides that contain a signal peptide. Using GFP labelling, the idi-1p was found to be located in the septa. idi-4 encodes a transcription factor containing a b-ZIP domain. The expresion of pspA, a gene coding for a subtilisine-like protease is also highly enhanced during incompatibility. We observed that the expression of most of these genes is also induced when incompatibility is released by the other non-allelic incompatibility system, het-C/het-E, but not by the allelic system het-s/het-S. Some of these genes are also induced under glucose and/or nitrogen starvation suggesting a functional relationship between the cell death reaction induced during starvation or by the coexpression of non-allelic het genes.

Translational control of gene expression.
Matthew S. Sachs, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute, Portland OR 97291-1000.

Short peptide coding regions (upstream open reading frames or uORFs) in the 5'-leaders of eukaryotic mRNAs can serve critical regulatory functions. uORFs are found in many mRNAs in filamentous fungi. A single uORF with an evolutionarily conserved peptide sequence is found upstream of the structural genes for the small subunit of arginine-specific carbamoyl phosphate synthetase from Neurospora crassa, Magnaporthe grisea, Trichoderma virens, Aspergillus nidulans and Saccharomyces cerevisiae. The N. crassa uORF specifies a 24-residue peptide named the arginine attenuator peptide (AAP) because it is involved in negative, Arg-specific translational regulation. In vivo, the N. crassa AAP down-regulates translation of ARG2 in response to Arg by reducing the average number of ribosomes associated with the arg-2 mRNA. AAP-mediated translational regulation has been reconstituted in an N. crassa cell-free translation system. A primer extension inhibition assay has been used to map the positions of ribosomes on capped and polyadenylated synthetic RNAs added to this system. Arg causes ribosomes to stall soon after they have translated the AAP. Arg-specific ribosome stalling is proposed to result in Arg-specific negative regulation because such ribosomes would block ribosomal scanning from the 5'-end of the mRNA and therefore block trailing ribosomes from translating ARG2. The AAP amino acid sequence, but not the RNA sequence encoding it, is critical for regulation. AAP translation can cause stalling of ribosomes involved in termination or elongation. Regulation by these evolutionarily conserved fungal leader peptides represent a novel mechanism of cis-acting translational control.

Chromatin modulation and regulation of pathogenic development in the smut fungus Ustilago maydis.
Joerg T. Kaemper, Michael Reichmann, Claudia Quadbeck-Seeger, and Regine Kahmann. Ludwig-Maximilian-University, Genetics, Munich, Bavaria, Germany.

In the phytopathogenic fungus Ustilago maydis the multiallelic b mating-type locus represents the central control locus for sexual and pathogenic development. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW that form heterodimers when originating from different alleles; the heterodimer is presumed to regulate pathogenicity genes, either directly by binding to cis regulatory sequences (class 1 genes), or indirectly via a b-dependent signal cascade (class 2 genes). Using a bE/bW fusion protein we were able to isolate the first direct target for the bE/bW heterodimer in the promoter of lga2, a gene located in the a locus. This sequence is bound by the bE/bW heterodimer and mediates the b-dependent regulation of the gene in vivo. In a screen for components of the b-dependent signal cascade we have isolated two different genes, one coding for a histone deacetylase (Hda1), the other one for a protein (Rum1) with similarities to the human retinoblastoma binding protein 2. Both genes are essential for the establishment of a repressed state of several class 2 genes in the absence of the b heterodimer. We propose that both Hda1 and Rum1 are in a complex with other proteins with at least one of them allowing sequence specific DNA binding. In this model, the regulation of gene activity is achieved by modulation of the chromatine structure mediated by the action of the histone deacetylase Hda1.

Genome defense in Neurospora.
Eric Selker, Joseph Dobosy, Michael Freitag, Shan Hays, Greg Kothe, Elena Kuzminova, Brian Margolin, Johanna Swanson and Hisashi Tamaru. Institute of Molecular Biology, University of Oregon, Eugene, OR 97405.

We wish to identify the important features of eukaryotic genomes and the forces responsible for shaping them. In organisms with small genomes, such as fungi, a large fraction of their DNA appears functional and may be maintained by natural selection alone. Even small genomes, however, are littered with sequences that look like selfish DNA. Although it is difficult to demonstrate that a particular sequence is useless, mechanisms well suited to fight selfish DNA have been found suggesting that genome structure is important. Neurospora has at least three distinct, but somewhat interrelated gene silencing systems that probably serve in genome defense: 1. RIP and RIP-associated methylation, 2. quelling, and 3. RIP-independent methylation of "foreign" DNA. We are using several approaches to investigate the control and mechanism of gene silencing systems - most particularly DNA methylation. One involves identification and characterization of mutants defective in methylation (dim). So far, we have identified four dim genes. Mutation of dim-2, which encodes a DNA methyltransferase, eliminates all methylation in vegetative cells and can derepress the transcription of some sequences and repress others. In the cases examined so far, methylation interferes with transcription elongation without affecting initiation. We have detected Neurospora proteins that bind to methylated sequences and may mediate these effects, potentially by influencing chromatin structure. Methylation-dependent silencing can be relieved by Trichostatin A (TSA), an inhibitor of histone deacetylases. Selective loss of methylation is associated with TSA treatment, suggesting that acetylation of chromatin proteins can directly or indirectly control DNA methylation. To explore the connection between DNA methylation and chromatin we are testing the effects of mutations in genes for histones H2A, H2B, H3, H4 and H1 and other candidate components of the methylation machine, including PCNA and a histone deacetylase.

Deletion of the gene encoding a typical histone H1 has no apparent phenotype in Aspegillus nidulans.
Ana Ramón, Ramón González, María Isabel Muro-Pastor, Irene García and Claudio Scazzocchio. Institut de Génétique et Microbiologie, Université Paris-Sud, URA D2225, 91405 Orsay Cedex, France.

A typical histone H1 can be purified from Aspergillus nidulans We have cloned, sequenced and deleted the gene (hhoA) coding for this protein. This gene comprises six introns. The position of one of the introns is identical to that found in a number of H1 plant histones. The peptidic sequence show the three typical domains including a recognisable globular domain. The closest similarity is found with the two globular domains of the H1-like protein of Saccahromyces cerevisiæ. Thus, the gene coding for the latter protein probably originated by internal duplication of the globular domain in a typical H1-coding gene. Deletion of hhoA results in complete disappearance of the H1 protein defined by electrophoretic mobility and PCA solubility. The deleted strain has no apparent phenotype. We have analysed growth, conidiation, conidial viability, UV and DMSO sensitivity, the appearance of resting and mitotic nuclei, the sexual cycle and ascospore viability. The nucleosomal repeat is identical in hhoA+ and hhoA- strains. We have analysed the nucleosomal structure of a number of promoters, including the niiA-niaD and prnD-prnB bidirectional promoters. In both expression is associated with important chromatin rearrangements The former shows complete nucleosome loss under conditions of full expression. This nucleosome loss is independent from transcription and strictly dependent on the activity of the GATA factor AreA. The deletion of hhoA does not change either the structure of the resting promoters nor the alterations associated with expression. The role of linker histones in fungi remains an open question.

Gene silencing, methylation and chromatin conformation in Ascobolus.
J. L. Barra 1,3 , G. Almouzni 2, L. Rhounim 1, G. Faugeron 1 and J.-L. Rossignol1. 1Institut Jacques Monod, Département de Microbiologie, UMR 7592: CNRS/Université Paris 7/Université Paris 6, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France. 2 Institut Curie/Section Recherche, UMR 144: CNRS/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. 3 Present address: Departamento de Química Biológica, Facultad de Ciencias Químicas, U. N. C., Ciudad Universitaria, 5000, Córdoba, Argentina.

The mechanisms by which cytosine methylation affects transcription are not completely elucidated. We took advantage of the process of methylation induced premeiotically (MIP) acting in Ascobolus to target in vivo methylation to different portions of two genes. Methylation of the promoters rarely led to gene silencing and inhibited transcription with variable efficiencies. In contrast, methylation of the coding sequences always led to gene silencing and resulted in the production of truncated transcripts with the length expected if methylation blocked transcript elongation. The analysis of the chromatin of the native met2 gene in unmethylated and methylated regions revealed that methylation led to a change in chromatin which was independent of the transcriptional state. The chromatin change was coextensive with methylation and started at the position where transcripts were truncated. These results suggest that the methylation-associated gene silencing process in Ascobolus could be mediated via a change in chromatin which would affect transcript elongation.

We also asked whether the linker histone H1 played a role in this in vivo methylation-associated chromatin modification observed in Ascobolus. It remains controversial whether histone H1 is also a methylated DNA-binding protein. We cloned the histone H1 gene from Ascobolus. We used MIP to create strains in which the native H1 gene was methylated. This resulted in its silencing as revealed by the complete disappearance of histone H1 protein, showing that H1 is not necessary for the MIP gene silencing process. The loss of histone H1 was associated with a hypermethylation of genomic DNA. In addition, strains lacking histone H1 stopped growing a few days after germination. The analysis of the chromatin of the met2 gene revealed that the methylation-associated chromatin modification was similar in strains in which the histone H1 gene was either active or silenced. These results provide a direct in vivo demonstration that histone H1 protein does not participate in methylation-associated chromatin modification in Ascobolus.

Inter-nuclear gene silencing in Phytophthora infestans.
Pieter van West1, Sophien Kamoun1,3, John W. van 't Klooster1, Neil A.R. Gow2, and Francine Govers. WAU, Phytopathology, Wageningen, The Netherlands. 2Univ of Aberdeen, Molecular Biology, Aberdeen, Scotland. 3 Present address: Department of Plant Pathology OSU, Wooster OH, USA.

Transformation of the diploid oomycete Phytophthora infestans with antisense, sense and promoter-less constructs of the coding sequence of the elicitin gene inf1 resulted in transcriptional silencing of both the transgenes and the endogenous gene. To investigate the mechanism of gene silencing we took advantage of the fact that P. infestans has coenocytic mycelia and that mycelial cells may contain multiple nuclei that can differ genetically, resulting in heterokaryotic strains. It appeared that: (i) transcriptional gene silencing is dominant in multinucleated cells, (ii) the silenced state can be transmitted from nucleus to nucleus in heterokaryotic strains, and (iii) gene silencing is maintained in a non-transformed nucleus after nuclear separation (van West et al., 1999). In addition, we showed that upon fusion of a silenced non-transgenic strain with a strain containing wildtype nuclei, the silenced state could be transmitted again to the wild type nuclei. Transcriptional gene silencing in P. infestans apparently involves inter-nuclear transfer of signals from silenced (transgenic and non-transgenic) nuclei to wild type nuclei, leading to stable gene silencing in the wild type nuclei. These findings support a model reminiscent of paramutation and involving a trans-acting factor that is capable of transferring a silencing signal between nuclei.


Light, mating types and other factors in regulation of development in Coprinus cinereus.
Ursula Kües, Alan P.F. Bottoli, Robert P. Boulianne, José D. Granado, Michaela J. Klaus, Simon Kuster, Yi Liu, Eline Polak, Piers J. Walser and M. Aebi. Inst. f. Mikrobiologie, ETH Zürich, Switzerland.

Coprinus cinereus alternates in its lifecycle between two mycelial stages, the monokaryon and the dikaryon with two genetic distinct nuclei per cell. Monokaryons form constitutively uninucleate asexual spores (oidia) within the aerial mycelium and may generate thickwalled chlamydospores and multicellular sclerotia. Dikaryons commonly form only few oidia, but abundant chlamydospores and sclerotia. Sclerotia develop from hyphal knots, areas of intense localized branching. Hyphal knots alternatively mature into fruitbody initials. Development of these different structures depends on the interplay between environmental signals such as light, temperature and nutrition, and internal factors such as the products of the mating type loci. The A mating type transcription factors play a central role in regulation. Blue light overrides A mediated repression of oidia production and A induced formation of chlamydospores, hyphal knots and sclerotia. At least in oidiation, the B mating type products modify the effect of A and light. In contrast to sclerotia formation, both A and light are needed to induce fruitbody initials. With fruitbody specific galectin genes we developed a read-out system to analyse the function of these and other regulators on regulation of fruitbody development. Currently, we study the role of RAS, cAMP and nutrition in fruiting. A collection of UV and REMI mutants helps us to unravel regulatory processes. Analysis of a mutant defective in the transversion of hyphal knot into fruitbody initials revealed a link to lipid metabolism.

The circadian system of Neurospora: A great model for the time of your life.
Jay C. Dunlap. Dartmouth Medical School, Biochemistry, Hanover, NH, USA.

Circadian rhythms are widespread among eukaryotes. Those molecularly understood appear to work in similar ways, and one of the best understood systems is that of Neurospora. The circadian clock appears to be a negative feedback loop wherein the frq gene, a known component of the clock in Neurospora crassa, encodes two FRQ proteins which travel to the nucleus to block the activity of the heterodimeric activator of frq comprised of the WC-1 and WC-2 proteins. Given appropriate delays in the synthesis, action, and turnover of FRQ, this negative feedback loop yields oscillations in frq transcript and FRQ protein levels; this cycling of clock components is characteristic of circadian oscillators. Light delivered at any point within the cycle acts rapidly through the WC proteins to increase the level of frq transcript thereby resetting the clock; mammalian circadian rhythms are reset in a similar manner. Temperature acts posttranscriptionally to determine the absolute level of FRQ in the cell and the site of initiation of translation within the frq transcript, thereby dictating the ratio of long FRQ versus short FRQ. Resetting of the clock by changes in temperature can be understood in terms of changes in these set points. More than a dozen circadianly expressed genes are known to act downstream of the clock. These clock-controlled genes (ccgs) include a hydrophobin (eas=ccg-2), trehalose synthase (ccg-9), and glyceraldehyde 3-P dehydrogenase (ccg-7=gpd) and play roles in clock regulation of development, stress responses, and intermediary metabolism.

WC-1 and WC-2 form a transcription complex that acts as a photoreceptor.
Giuseppe Macino, Paola Ballario, Claudio Talora, Lisa Franchi, Hartmut Linden Universita' di Roma La Sapienza.

The filamentous fungus Neurospora crassa is currently considered an important model for the study of blue light signal transduction, owing to the simple genetics of the pleiotropic responses to blue light stimuli. The two central components of blue light response, White Collar-1(WC-1) and White Collar-2 (WC-2), appear to encode zinc-finger putative transcription factors, essential for light activated transcription (Ballario et al., 1996; Linden and Macino, 1997). The products of the wc genes are required for all processes that are under control of blue light such as mycelial carotenogenesis, circadian rythm of conidiation, and phototropism of perithecial beaks (for a review Ballario and Macino, 1998 and Linden et al., 1998). PAS, the multifunctional domain found in eubacteria, archaebacteria and eukaryotes involved in dimerization (Ponting and Aravind, 1997 ) is present in both WC-1 and WC-2. Furthermore a PAS degenerated domain called LOV (for Light, Oxygen and Voltage), identified in prokaryotic proteins (like PYP, Bat, and NifL ) and the plant blue light photoreceptor NPH1 (Huala et al., 1998), where it seems to act as a versatile flavin-binding domain, is present in WC-1. The in vitro ability of the White Collar proteins to form hetero and homodimers via their PAS domain has been demostrated (Ballario et al., 1998). Now we report the biochemical analysis by antibodies immunodetection of the proteins WC-1 and WC-2 under dark and light growth conditions. WC-1 and WC-2 are both present in the dark. Their fate, upon light irradiation, has been investigated in wild type Neurospora strains and in wc-2 and wc-1 genetic backgrounds. We show that WC-1 and WC-2, undergoes a light-induced hyperphosphorylation. We observe by coimmunoprecipitation the presence of a WC-1 WC-2 complex (WCC) in the dark and in the light. A new model of Neurospora blue light transduction, based on WCC complex phosphorylation is presented.

The fluffy gene of Neurospora crassa encodes a Cys6-Zn2 cluster protein that regulates macroconidiation.
Daniel J. Ebbole, and Lori Bailey Shrode. Texas A&M; University, Plant Pathol. & Microbiol, College Station, Texas, USA.

Macroconidiation provides N. crassa with a means to efficiently and rapidly disperse itself. Fluffy (fl) is a regulator of macroconidiation and encodes a member of the Gal4 class of transcription factors. Null mutations of fl block the switch from filamentous growth to the budding growth characteristic of proconidial chain formation. These mutants are also blocked in expression of many of the known conidiation-specific genes. The pattern of fl mRNA expression is consistent with its role as a regulator of morphogenesis. A basal level of fl expression is observed in undifferentiated mycelia. fl mRNA levels are induced during development at approximately the time when budding growth initiates and fl mRNA levels declines at later stages of development. acon-2 and acon-3 also are regulators of conidial morphogenesis and acon-2 is required for induction of fl mRNA while acon-3 is not. This finding is consistent with the view that acon-2 precedes fl in the pathway regulating conidiophore development and that acon-3 functions at the same time as fl or later. Elevated expression of fl from a constitutive promoter was sufficient to induce conidiophore morphogenesis in minimal medium. However, not all conidiation-induced genes were expressed. Although fl is necessary and sufficient to induce conidiophore morphogenesis, additional factors are required to coordinate activation of some of the conidiation-induced genes.

Analysis of an opsin gene from Neurospora crassa.
Jennifer A. Bieszke1, Katherine A. Borkovich1, Donald O. Natvig2, Laura E. Bean2, Edward L. Braun3, and Seogchan Kang4. 1University of Texas Medical School, Micro. and Mol. Genetics, Houston, TX, USA. 2Univ. of New Mexico, Department of Biology, Albuquerque, NM, USA. 3Ohio State University, Dept. Plant Biology, Columbus, OH, USA. 4Pennsylvania State Univ., Dept. Plant Pathology, University Park, PA, USA.

Opsins are a class of retinal-binding, seven-helix transmembrane proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had only been identified in animals and the archaea. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The amino acid sequence of nop-1 predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues. Furthermore, NOP-1 contains the conserved lysine residue that forms a Schiff base linkage with retinal in other rhodopsins, and two acidic residues essential for H+ transport in bacteriorhodopsin. Evolutionary analysis revealed clear relatedness between NOP-1 and archaeal opsins, as well as between NOP-1 and several fungal opsin-related proteins. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Functional residues conserved between NOP-1 and archaeal opsins suggest a role for NOP-1 in photobiology. Results from Northern analysis support conidiation and light-based regulation of nop-1 gene expression. delta nop-1 strains exhibit a synthetic light-dependent effect upon conidiation in the presence of the mitochondrial H+-ATPase inhibitor oligomycin. We propose that eukaryotic opsins represent a relatively ancient group of proteins involved in light perception.


A Protein Pathogenicity Factor from Pyrenophora tritici-repentis
Linda Ciuffetti. Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.

Pyrenophora tritici-repentis is the causal agent of tan spot of wheat and known to be a destructive pathogen worldwide. Certain isolates of the fungus have been shown to produce in culture a host-specific toxin(s) (HST) that induces typical tan spot necrosis upon infiltration into tissue of susceptible wheat cultivars. Purification of the major necrosis-inducing toxin (Ptr ToxA) enabled us to clone the gene (ToxA) for this HST. Fungal transformation studies confirmed that this gene functions in the plant as a primary determinant of pathogenicity in the Pyrenophora-wheat interaction.

The ToxA open reading frame (ORF) encodes a preproprotein (19.7 kD), with an N-terminal signal peptide, followed by a dual domain (N + C) protoxin. Analysis of the mature Ptr ToxA by mass spectroscopy indicated a size of 13.2 kD. Additional proteolytic processing is involved in the production of the mature Ptr ToxA. Treatment of the mature toxin with the enzyme pyroglutamate amino peptidase confirmed the amino-terminal position of Ptr ToxA to be amino acid residue Gln-61.

Following purification of Ptr ToxA and the cloning of the ToxA gene, we have recently directed our efforts toward the elucidation of the site- and mode-of-action of this toxin in sensitive wheat. One approach to investigate possible protein-protein interactions between Ptr ToxA and sensitive wheat, is a genetical approach utilizing the yeast two-hybrid system. The cDNAs of a toxin-sensitive wheat cultivar were directionally cloned into a lambda vector. Following extensive screening of the library for positive interacting proteins, we identified a cDNA clone with a coding sequence of 426bp. Database searches did not reveal any significant homology to identified proteins. Attempts to identify the complete ORF of this putative interacting protein are in progress. Additionally, labeled derivatives of the toxin have been produced that should be useful for a biochemical approach to the identification of the toxin's site-of-action. To produce biologically active derivatives of Ptr ToxA that could be used for receptor-binding experiments, functional Ptr ToxA was expressed and purified from Escherichia coli. Polyhistidine-tagged, fusion protein (NC-FP) consists of both the N- and C-domains of the ToxA ORF and elicits cultivar-specific necrosis in sensitive wheat genotypes, with a specific activity similar to native toxin. A fusion protein consisting of the C-domain only is far less active (ca. one-fifth that of the native protein). These studies indicate that the N-domain is necessary for efficient folding of toxin and that post-translational modifications of Ptr ToxA are not essential for activity. Labeled NC-FP retains significant activity as compared to the unmodified NC-FP. Labeled NC-FP is currently being utilized in both in vitro and in vivo binding assays to identify potentially interacting wheat proteins. Comparison of the results from the yeast-two hybrid screen and the binding assays with labeled NC-FP will potentially identify wheat proteins that play a significant role in the signal transduction pathway of this host-pathogen interaction.

A pathogenicity island in Nectria haematococca.
H. Corby Kistler1, Yinong Han1, Ulla Benny1, Xiaoguang Liu2, Esteban Temporini2, Hans VanEtten2. 1University of Florida, Gainesville, FL USA; 2University of Arizona, Tucson, AZ USA.

Host range determinants of fungal plant pathogens are poorly understood. In some instances it appears that the ability to cause disease on a particular host plant species is determined by a single gene with large effect (e.g. the avenacinse gene from Gaeumannomyces graminis, PWL2 from Magnaporthe grisea). However in Nectria haematococca MPVI, pathogenicity to pea is conferred by a cluster of genes, each individually having a small but significant effect. These "PEP" genes are within 25 kb of each other and located on a conditionally dispensable (CD) chromosome. The PEP gene cluster contains six genes that are expressed during infection of pea tissue but the biochemical function of only one of the genes is known with certainty. This gene, PDA1, encodes a specific cytochrome P450 that confers resistance to pisatin, an antibiotic produced by pea plants. Three of the PEP genes, in addition to PDA1, can independently confer some level of virulence to an isolate lacking the CD chromosome; functions for two of these three genes are hypothesized, based on predicted amino acid sequences. The deduced amino acid sequence of another transcribed portion of the PEP cluster, as well as three apparently non-transcribed open reading frames, have a high degree of similarity to known fungal transposases. Both the G+C content and codon usage of the six genes in the PEP cluster differ from that of genes on other N. haematococca chromosomes. Several of the features of the PEP cluster - a cluster of pathogenicity genes, the presence of transposable elements and suggestions of exogenous origin - are shared by pathogenicity islands in pathogenic bacteria of plants and animals.

Melanin and Fe(II) as an extracellular redox buffering system.
Eric S. Jacobson. McGuire Veterans Affairs Medical Center, Richmond, Virginia USA , and Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, USA.

Melanin is a fungal extracellular redox buffer which, in principle, can neutralize antimicrobial oxidants generated by immunologic effector cells, but its source of reducing equivalents is not known. We wondered whether the large quantities of Fe(II) generated by the external ferric reductase of fungi might have the physiologic function of reducing fungal melanin and thereby promoting pathogenesis. We observed that exposure of a melanin film electrode to reductants decreased the open-circuit potential and reduced the area of a cyclic voltammetric reduction wave, whereas exposure to oxidants produced the opposite effects. Exposure to 10, 100, 1,000 or 10,000 µM Fe(II) decreased the open circuit potential of melanin by 0.015, 0.038, 0.100 and 0.120 V, respectively, relative to a silver-silver chloride standard, and decreased the area of the cyclic voltammetric reduction wave by 27, 35, 50 and 83%, respectively. Moreover, exposure to Fe(II) increased the buffering capacity by 44%, while exposure to millimolar dithionite did not increase the buffering capacity. The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffering. Light absorption by melanin suspensions was decreased 14% by treatment with Fe(II), consistent with reduction of melanin. Light absorption by suspensions of melanized Cryptococcus neoformans was decreased 1.3% by treatment with Fe(II) (P < 0.05). Cultures of C. neoformans generated between 2 and 160 µM Fe(II) in cultural supernatants, depending upon the strain and the conditions [the higher values were achieved by a constitutive ferric reductase mutant in high concentrations of Fe (III)]. We infer that Fe(II) can reduce melanin under physiologic conditions; moreover, it binds to melanin and cooperatively increases redox buffering. The data support a model for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular Fe(II) maintain the reducing capacity of the extracellular redox buffer.

Session IV: CELL BIOLOGY Chair:Walter Neupert

Motors and intracellular organelle movements.
Gero Steinberg. Ludwig-Maximilians-Universitaet, Institut fuer Genetik, Muenchen, Germany.

Polarized secretion and intracellular transport of membranous organelles are crucial requirements for fungal growth and morphogenesis. Organelle movements are mediated by fibrous elements of the cytoskeleton, which serve as tracks for mechanoenzymes that convert chemical energy into motion. Recently, several fungal representatives of microtubule-dependent kinesins and dyneins, as well as several actin-based myosins were identified, and genetic and cell biological investigations greatly extended our knowledge of their biological role. However, most work was done on the yeast S. cerevisiae, and besides nuclear migration our understanding of the molecular machinery for long distance transport of membranous organelles in dimorphic and filamentous fungi is still fragmentary. Therefore, we started to study the biological role of several potential organelle motors of the kinesin, dynein and myosin protein family in the dimorphic plant pathogen Ustilago maydis. Our data indicate a role of dynein and myosin in nuclear migration and secretion, respectively, which is in agreement with findings for S. cerevisiae. However, in contrast to yeast, microtubules play a central role in polarized secretion, endocytosis and tip growth of U. maydis sporidia and hyphae. The central role of the microtubule cytoskeleton is confirmed by the existence of two kinesin motors that are conserved among higher eukaryotes but are absent from S. cerevisiae. The existence of a complex microtubule cytoskeleton establishes U. maydis as a simple model system for cytoskeleton analysis.

Novel proteins required for nuclear distribution in Neurospora crassa.
Michael Plamann1, In Hyung Lee1, Santosh Kumar1, Peter Minke2, and John Tinsley2. University of Missouri-Kansas City, School of Biol. Sciences, Kansas City, MO, USA. 2Texas A&M; University, Biology, College Station, TX, USA.

Cytoplasmic dynein is the most complex of the cytoplasmic microtubule-associated motor proteins, and it has been shown to be required for the movement and positioning of nuclei in fungi. We have developed a genetic system for the analysis of cytoplasmic dynein in the filamentous fungus Neurospora crassa. We have shown previously that the N. crassa ro-1, ro-3, and ro-4 genes encode subunits of either cytoplasmic dynein or dynactin, a dynein-associated complex. We have isolated hundreds of ro mutants, and the availability of multiple independent alleles of ro genes encoding known subunits of cytoplasmic dynein and dynactin provide us with the opportunity to do a detailed analysis of protein interactions and specific functions within the motor complex. We present our analysis of a specific ro-4 allele and our initial analysis of various ro-1 and ro-3 alleles. We also report that five additional ro genes encode novel proteins. ro-7 is predicted to encode a 70 kD protein distantly related to actin. In ro-7 mutants, cytoplasmic dynein and the dynactin complex accumulate at spindle pole bodies suggesting that RO7 is required for proper intracellular targeting of cytoplasmic dynein and dynactin. ro-10 is predicted to encode a novel 24 kD protein that may be required for stability of the dynactin complex, because p150Glued,the largest subunit of the dynactin complex, is not detectable in a ro-10 deletion strain. Our results suggest that at least some of these novel proteins are required for the proper function of cytoplasmic dynein or dynactin.

Protein import into mitochondria.
M. Brunner, S. Nussberger, D. Rapaport, R.A. Stuart, and W. Neupert. Institut fur Physiologische Chemie, Universitat Munchen, Goethestr. 33, D-80336 Munchen.

The majority of mitochondrial proteins are encoded by the nucleus, are synthesized in the cytoplasm as preproteins and are imported in a post-translational manner into the mitochondria. Transport of proteins into the mitochondria is catalyzed by protein translocation machineries located in both the outer and inner mitochondrial membranes, the TOM and the TIM complexes, respectively.
The TOM complex mediates the recognition of preproteins, their transfer through the outer membrane and the insertion of resident outer membrane proteins. We have recently purified the TOM complex from N. crassa. Reconstitution studies shows it forms cation selective, high conductance channels. Electron microscopy revealed the isolated TOM complex particles are about 138 in diameter and appear to contain two or three pores per particle. Further translocation across the inner membrane requires a membrane potential and is mediated by one, of at least two, import machineries, the TIM complexes. Translocation of presequence-targeted proteins is facilitated by the Tim17-Tim23 machinery, which operates closely with Tim44, mt-Hsp70 and Mge1p, to drive import into the matrix in an ATP-dependent manner.
A second independent, translocation machinery, termed the Tim22-Tim54 translocase mediates the insertion of members of the mitochondrial carrier family into the inner membrane. Three related proteins, of the intermembrane space, Tim9, Tim10 and Tim12, small metal binding proteins, interact directly with the incoming carrier proteins at the TOM complex, to mediate their passage through the intermembrane space. Together with Tim22-Tim54, Tim9, Tim10 and Tim12 facilitate the insertion of the carrier proteins directly into the inner membrane. Finally, a subset of inner membrane proteins, some nuclear encoded, others mitochondrial encoded, reach their correct orientation in the inner membrane, via a membrane potential-dependent export step from the matrix. We have recently identified a novel translocase in the inner membrane, the Oxa1p complex, which mediates this export step.
Adam, A., Endres, M., Sirrenberg, C., Lottspeich, F., Neupert, W., and Brunner, M. (1999). Tim9, a new component of the Tim22-54 translocase in mitochondria. EMBO J. 18, 313-319.
Hell, K., Hermann, J. M., Pratje, E., Neupert, W., and Stuart, R. A. (1997). Oxa1p mediates the export of the N- and C-termini of preCoxII from the mitochondrial matrix to the intermembrane space. FEBS Lett. 418, 367-370.
Hell, K., Herrmann, J. M., Pratje, E., Neupert, W., and Stuart, R. A. (1998). Oxa1p, an essential component of the N-tail protein export machinery in mitochondria. Proc. Natl. Acad. Sci. USA 95, 2250-2255.
Kunkele, K.-P., Heins, S., Dembowski, M., Nargang, F. E., Benz, R., Thieffry, M., Walz, J., Lill, R., Nussberger, S., and Neupert, W. (1998). The preprotein translocation channel of the outer membrane of mitochondria. Cell 93, 1009-1019.
Neupert, W. (1997). Protein import into mitochondria. Annu. Rev. Biochem. 66, 863-917. Sirrenberg, C., Bauer, M. F., Guiard, B., Neupert, W., and Brunner, M. (1996). Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature 384, 582-585.
Sirrenberg, C., Endres, M., Folsch, H., Stuart, R. A., Neupert, W., and Brunner, M. (1998). Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/mrs5. Nature 391, 912-915.

Mitochondrial fusion and the transmembrane GTPase, FZO1P.
Greg J. Hermann1, John W. Thatcher1, John P. Mills2, Karen G. Hales2, Margaret T. Fuller2, Jodi Nunnari3 and Janet M. Shaw1 1Department of Biology, University of Utah, Salt Lake City, UT; 2Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA; 3Department of Molecular and Cellular Biology, University of California, Davis, CA.

Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (Fzo) GTPase block a developmentally-regulated mitochondrial fusion event during spermatogenesis (Hales, K. G. and M. T. Fuller. 1997. Cell. 80:121-129). The predicted fuzzy onions protein belongs to a novel family of high molecular weight GTPases also present in yeast, nematodes, and mammals. We have shown that the yeast ortholog of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.