The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.

Cephalosporins as key lead generation beta-lactam antibiotics.

Antibiotics are antibacterial compounds that interfere with bacterial growth, without harming the infected eukaryotic host. Among the clinical agents, beta-lactams play a major role in treating infected humans and animals. However, the ever-increasing antibiotic resistance crisis is forcing the pharmaceutical industry to search for new antibacterial drugs to combat a range of current and potential multi-resistant bacterial pathogens. In this review, we provide an overview of the development, innovation, and current status of therapeutic applications for beta-lactams with a focus on semi-synthetic cephalosporins. Cephalosporin C (CPC), which is a natural secondary metabolite from the filamentous fungus Acremonium chrysogenum, plays a major and demanding role in both producing modern antibiotics and developing new ones. CPC serves as a core compound for producing semi-synthetic cephalosporins that can control infections with different resistance mechanisms. We therefore summarize our latest knowledge about the CPC biosynthetic pathway and its regulation in the fungal host. Finally, we describe how CPC serves as a key lead generation source for the in vitro and better, in vivo synthesis of 7-aminocephalosporanic acid (7-ACA), the major core compound for the pharmaceutical synthesis of current and future semi-synthetic cephalosporins. KEY POINTS: • Latest literature on cephalosporin generations • Biotechnical production of cephalosporins • In vivo production of 7-ACA.

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth.

The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

STRIPAK, a highly conserved signaling complex, controls multiple eukaryotic cellular and developmental processes and is linked with human diseases.

The striatin-interacting phosphatases and kinases (STRIPAK) complex is evolutionary highly conserved and has been structurally and functionally described in diverse lower and higher eukaryotes. In recent years, this complex has been biochemically characterized better and further analyses in different model systems have shown that it is also involved in numerous cellular and developmental processes in eukaryotic organisms. Further recent results have shown that the STRIPAK complex functions as a macromolecular assembly communicating through physical interaction with other conserved signaling protein complexes to constitute larger dynamic protein networks. Here, we will provide a comprehensive and up-to-date overview of the architecture, function and regulation of the STRIPAK complex and discuss key issues and future perspectives, linked with human diseases, which may form the basis of further research endeavors in this area. In particular, the investigation of bi-directional interactions between STRIPAK and other signaling pathways should elucidate upstream regulators and downstream targets as fundamental parts of a complex cellular network.

AcAxl2 and AcMst1 regulate arthrospore development and stress resistance in the cephalosporin C producer Acremonium chrysogenum.

The filamentous fungus Acremonium chrysogenum is the primordial producer of the ß-lactam antibiotic cephalosporin C. This antibiotic is of major biotechnological and medical relevance because of its antibacterial activity against Gram-positive and Gram-negative bacteria. Antibiotic production during the lag phase of fermentation is often accompanied by a typical morphological feature of A. chrysogenum, the fragmentation of the mycelium into arthrospores. Here, we sought to identify factors that regulate the hyphal septation process and present the first comparative functional characterization of the type I integral plasma membrane protein Axl2 (axial budding pattern protein 2), a central component of the bud site selection system (BSSS) and Mst1 (mammalian Sterile20-like kinase), a septation initiation network (SIN)-associated germinal center kinase (GCK). Although an Acaxl2 deletion strain showed accelerated arthrospore formation after 96 h in liquid culture, deletion of Acmst1 led to a 24 h delay in arthrospore development. The overexpression of Acaxl2 resulted in an arthrospore formation similar to the A3/2 strain. In contrast to this, A3/2::Acmst1 OE strain displayed an enhanced arthrospore titer. Large-scale stress tests revealed an involvement of AcAxl2 in controlling osmotic, endoplasmic reticulum, and cell wall stress response. In a similar approach, we found that AcMst1 plays an essential role in regulating growth under osmotic, cell wall, and oxidative stress conditions. Microscopic analyses and plating assays on media containing Calcofluor White and NaCl showed that arthrospore development is a stress-dependent process. Our results suggest the potential for identifying candidate genes for strain improvement programs to optimize industrial fermentation processes.

The transcription factor PRO44 and the histone chaperone ASF1 regulate distinct aspects of multicellular development in the filamentous fungus Sordaria macrospora.

BACKGROUND: Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail. RESULTS: The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between Δasf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in Δasf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of Δpro44, results in defects during ascospore maturation. CONCLUSIONS: In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi.

In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications.

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns.

In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaA-i2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatography-mass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii.

Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity.

Transcriptome analysis of the two unrelated fungal ß-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs.

BACKGROUND: Cephalosporins and penicillins are the most frequently used ß-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains. RESULTS: Using the sequence of PcbC, a key enzyme of ß-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for ß-lactam production. Other genes not essential for ß-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains. We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including ß-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains. CONCLUSIONS: The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal ß-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved ß-lactam antibiotic titers during strain improvement in both fungi.

Transcription factor PRO1 targets genes encoding conserved components of fungal developmental signaling pathways.

The filamentous fungus Sordaria macrospora is a model system to study multicellular development during fruiting body formation. Previously, we demonstrated that this major process in the sexual life cycle is controlled by the Zn(II)2 Cys6 zinc cluster transcription factor PRO1. Here, we further investigated the genome-wide regulatory network controlled by PRO1 by employing chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to identify binding sites for PRO1. We identified several target regions that occur in the promoter regions of genes encoding components of diverse signaling pathways. Furthermore, we identified a conserved DNA-binding motif that is bound specifically by PRO1 in vitro. In addition, PRO1 controls in vivo the expression of a DsRed reporter gene under the control of the esdC target gene promoter. Our ChIP-seq data suggest that PRO1 also controls target genes previously shown to be involved in regulating the pathways controlling cell wall integrity, NADPH oxidase and pheromone signaling. Our data point to PRO1 acting as a master regulator of genes for signaling components that comprise a developmental cascade controlling fruiting body formation.

Deactivation of the autotrophic sulfate assimilation pathway substantially reduces high-level ß-lactam antibiotic biosynthesis and arthrospore formation in a production strain from Acremonium chrysogenum.

The filamentous ascomycete Acremonium chrysogenum is the only industrial producer of the ß-lactam antibiotic cephalosporin C. Synthesis of all ß-lactam antibiotics starts with the three amino acids l-α-aminoadipic acid, l-cysteine and l-valine condensing to form the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of l-cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the Acr. chrysogenum genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either l-cysteine or l-homocysteine, and a reverse transsulfuration pathway, where l-methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both l-methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from Acr. chrysogenum.

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

BACKGROUND: Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. RESULTS: We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. CONCLUSIONS: Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

New insights from an old mutant: SPADIX4 governs fruiting body development but not hyphal fusion in Sordaria macrospora.

During the sexual life cycle of filamentous fungi, multicellular fruiting bodies are generated for the dispersal of spores. The filamentous ascomycete Sordaria macrospora has a long history as a model system for studying fruiting body formation, and two collections of sterile mutants have been generated. However, for most of these mutants, the underlying genetic defect remains unknown. Here, we investigated the mutant spadix (spd) that was generated by X-ray mutagenesis in the 1950s and terminates sexual development after the formation of pre-fruiting bodies (protoperithecia). We sequenced the spd genome and found a 22 kb deletion affecting four genes, which we termed spd1-4. Generation of deletion strains revealed that only spd4 is required for fruiting body formation. Although sterility in S. macrospora is often coupled with a vegetative hyphal fusion defect, Δspd4 was still capable of fusion. This feature distinguishes SPD4 from many other regulators of sexual development. Remarkably, GFP-tagged SPD4 accumulated in the nuclei of vegetative hyphae and fruiting body initials, the ascogonial coils, but not in sterile tissue from the developing protoperithecium. Our results point to SPD4 as a specific determinant of fruiting body formation. Research on SPD4 will, therefore, contribute to understanding cellular reprogramming during initiation of sexual development in fungi.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

A MAT1-2 wild-type strain from Penicillium chrysogenum: functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain.

In heterothallic ascomycetes, mating is controlled by two nonallelic idiomorphs that determine the 'sex' of the corresponding strains. We recently discovered mating-type loci and a sexual life cycle in the penicillin-producing fungus, Penicillium chrysogenum. All industrial penicillin production strains worldwide are derived from a MAT1-1 isolate. No MAT1-2 strain has been investigated in detail until now. Here, we provide the first functional analysis of a MAT1-2 locus from a wild-type strain. Similar to MAT1-1, the MAT1-2 locus has functions beyond sexual development. Unlike MAT1-1, the MAT1-2 locus affects germination and surface properties of conidiospores and controls light-dependent asexual sporulation. Mating of the MAT1-2 wild type with a MAT1-1 high penicillin producer generated sexual spores. We determined the genomic sequences of parental and progeny strains using next-generation sequencing and found evidence for genome-wide recombination. SNP calling showed that derived industrial strains had an uneven distribution of point mutations compared with the wild type. We found evidence for meiotic recombination in all chromosomes. Our results point to a strategy combining the use of mating-type genes, genetics, and next-generation sequencing to optimize conventional strain improvement methods.

Genome-wide identification of target genes of a mating-type α-domain transcription factor reveals functions beyond sexual development.

Penicillium chrysogenum is the main industrial producer of the ß-lactam antibiotic penicillin, the most commonly used drug in the treatment of bacterial infections. Recently, a functional MAT1-1 locus encoding the α-box transcription factor MAT1-1-1 was discovered to control sexual development in P. chrysogenum. As only little was known from any organism about the regulatory functions mediated by MAT1-1-1, we applied chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to gain new insights into the factors that influence MAT1-1-1 functions on a molecular level and its role in genome-wide transcriptional regulatory networks. Most importantly, our data provide evidence for mating-type transcription factor functions that reach far beyond their previously understood role in sexual development. These new roles include regulation of hyphal morphology, asexual development, as well as amino acid, iron, and secondary metabolism. Furthermore, in vitro DNA-protein binding studies and downstream analysis in yeast and P. chrysogenum enabled the identification of a MAT1-1-1 DNA-binding motif, which is highly conserved among euascomycetes. Our studies pave the way to a more general understanding of these master switches for development and metabolism in all fungi, and open up new options for optimization of fungal high production strains.

The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling.

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing.

The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors.

A fungal sarcolemmal membrane-associated protein (SLMAP) homolog plays a fundamental role in development and localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria.

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.