Hans-Peter Schmitz

Evolution of multinucleated Ashbya gossypii hyphae from a budding yeast-like ancestor.

In the filamentous ascomycete Ashbya gossypii polarity establishment at sites of germ tube and lateral branch emergence depends on homologues of Saccharomyces cerevisiae factors controlling bud site selection and bud emergence. Maintenance of polar growth involves homologues of well-known polarity factors of budding yeast. To achieve the much higher rates of sustained polar surface expansion of hyphae compared to mainly non-polarly growing yeast buds five important alterations had to evolve. Permanent presence of the polarity machinery at a confined area in the rapidly expanding hyphal tip, increased cytoplasmic space with a much enlarged ER surface for generating secretory vesicles, efficient directed transport of secretory vesicles to and accumulation at the tip, increased capacity of the exocytosis system to process these vesicles, and an efficient endocytosis system for membrane and polarity factor recycling adjacent to the zone of exocytosis. Morphological, cell biological, and molecular aspects of this evolution are discussed based on experiments performed within the past 10 y.

Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions.

Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly inxa0vitro, we wanted to test this phenomenon under inxa0vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V1 subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V1 and Vo even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.

The function of two closely related Rho proteins is determined by an atypical switch I region

We show here that the encoded proteins of the two duplicated RHO1 genes from the filamentous fungus Ashbya gossypii, AgRHO1a and AgRHO1b have functionally diverged by unusual mutation of the conserved switch I region. Interaction studies and in vitro assays suggest that a different regulation by the two GTPase activating proteins (GAPs) AgLrg1 and AgSac7 contributes to the functional differences. GAP-specificity and protein function is determined to a large part by a single position in the switch I region of the two Rho1 proteins. In AgRho1b, this residue is a tyrosine that is conserved among the Rho-protein family, whereas AgRho1a carries an atypical histidine at the same position. Mutation of this histidine to a tyrosine changes GAP-specificity, protein function and localization of AgRho1a. Furthermore, it enables the mutated allele to complement the lethality of an AgRHO1b deletion. In summary, our findings show that a simple mutation in the switch I region of a GTP-binding protein can change its affinity towards its GAPs, which finally leads to a decoupling of very similar protein function without impairing effector interaction.

Milk and sugar: Regulation of cell wall synthesis in the milk yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis is an alternative model yeast to the well established Saccharomyces cerevisiae. The cell wall of these fungi consists of polysaccharides (i.e. long chains of β-1,3- and β-1,6-linked sugar chains and some chitin) and mannoproteins, both of which are continually adapted to environmental conditions in terms of their abundance and organization. This implies the need to perceive signals at the cell surface and to transform them into a proper cellular response. The signal transduction cascade involved in this process is generally referred to as the cell wall integrity (CWI) pathway. CWI signaling and cell wall composition have been extensively studied in the Bakers yeast S. cerevisiae and are also of interest in other yeast species with commercial potential, such as K. lactis. We here summarize the results obtained in the past years on CWI signaling in K. lactis and use a comparative approach to the findings obtained in S. cerevisiae to highlight special adaptations to their natural environments.

Identification of Dck1 and Lmo1 as upstream regulators of the small GTPase Rho5 in Saccharomyces cerevisiae

The exact function and regulation of the small GTPase Rho5, a putative homolog of mammalian Rac1, in the yeast Saccharomyces cerevisiae have not yet been elucidated. In a genetic screen initially designed to identify novel regulators of cell wall integrity signaling, we identified the homologs of mammalian DOCK1 (Dck1) and ELMO (Lmo1) as upstream components which regulate Rho5. Deletion mutants in any of the encoding genes (DCK1, LMO1, RHO5) showed hyper‐resistance to cell wall stress agents, demonstrating a function in cell wall integrity signaling. Live‐cell fluorescence microscopy showed that Dck1, Lmo1 and Rho5 quickly relocate to mitochondria under oxidative stress and cell viability assays indicate a role of Dck1/Lmo1/Rho5 signaling in triggering cell death as a response to hydrogen peroxide treatment. A regulatory role in autophagy/mitophagy is suggested by the colocalization of Rho5 with autophagic markers and the decreased mitochondrial turnover observed in dck1, lmo1 and rho5 deletion mutants. Rho5 activation may thus serve as a central hub for the integration of different signaling pathways.

A Bnr‐like formin links actin to the spindle pole body during sporulation in the filamentous fungus Ashbya gossypii

Formin proteins are nucleators of actin filaments and regulators of the microtubule cytoskeleton. As such, they play important roles in the development of yeast and other fungi. We show here that AgBnr2, a homologue of the ScBnr1 formin from the filamentous fungus Ashbya gossypii, localizes to the spindle pole body (SPB), the fungal analogue of the centrosome of metazoans. This protein plays an important role in the development of the typical needle‐shaped spores of A. gossypii, as suggested by several findings. First, downregulation of AgBNR2 causes defects in sporangium formation and a decrease in the total spore number. Second, a fusion of AgBNR2 to GFP that is driven by the native AgBNR2 promoter is only visible in sporangia. Third, AgBnr2 interacts with a AgSpo21, a sporulation‐specific component of the SPB. Furthermore, we provide evidence that AgBnr2 might nucleate actin cables, which are connected to SPBs during sporulation. Our findings add to our understanding of fungal sporulation, particularly the formation of spores with a complex, elongated morphology, and provide novel insights into formin function.

The small GTP-binding proteins AgRho2 and AgRho5 regulate tip-branching, maintenance of the growth axis and actin-ring-integrity in the filamentous fungus Ashbya gossypii.

GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.

A network involving Rho-type GTPases, a paxillin and a formin homologue regulates spore length and spore wall integrity in the filamentous fungus Ashbya gossypii.

Fungi produce spores that allow for their dispersal and survival under harsh environmental conditions. These spores can have an astonishing variety of shapes and sizes. Using the highly polar, needle‐shaped spores of the ascomycete Ashbya gossypii as a model, we demonstrated that spores produced by this organism are not simple continuous structures but rather consist of three different segments that correlate with the accumulation of different materials: a rigid tip segment, a more fragile main spore‐compartment and a solid tail segment. Little is currently known about the regulatory mechanisms that control the formation of the characteristic spore morphologies. We tested a variety of mutant strains for their spore phenotypes, including spore size, shape and wall defects. The mutants that we identified as displaying such phenotypes are all known for their roles in the regulation of hyphal tip growth, including the formin protein AgBni1, the homologous Rho‐type GTPases AgRho1a and AgRho1b and the scaffold protein AgPxl1. Our observations suggest that these proteins form a signalling network controlling spore length by regulating the formation of actin structures.

Selection of STOP-free sequences from random mutagenesis for 'loss of interaction' two-hybrid studies.

The investigation of protein–protein interactions is an essential part of biological research. To obtain a deeper insight into regulatory protein networks, the identification of the components, domains and especially single residues that are involved in these interactions is helpful. A widespread and attractive genetic tool for investigation of protein–protein interactions is the yeast two‐hybrid system. This method enables large‐scale screens and its application is cheap and relatively simple. For identification of the amino acids in a protein sequence that are essential for interaction with a specific partner, yeast two‐hybrid assays can be combined with random mutagenesis of the sequence of interest. A common problem with such an experiment is the generation of stop codons within the mutagenized fragments, leading to the isolation of many false positives when screening for loss of interaction using the two‐hybrid method. To overcome this problem, we modified the yeast two‐hybrid system to allow selection for sequences without stop codons. To achieve this, we fused the ScURA3 marker‐gene in frame to the mutagenized fragments. We show here that this marker is fully functional when fused to a two‐hybrid construct with a nuclear localization signal, such as a Gal4 activation domain and a prey protein, thus allowing selection of stop‐free sequences on media without uracil. Using the Rho‐binding domain from a Bni1‐like formin and different Rho‐type GTPases from Ashbya gossypii as examples, we further show that our system can be used to screen large numbers of transformants for loss of protein–protein interactions in combination with random mutagenesis. Copyright

Septin-associated protein kinase Gin4 affects localization and phosphorylation of Chs4, the regulatory subunit of the Baker’s yeast chitin synthase III complex

Chitin is mainly formed by the chitin synthase III complex (CSIII) in yeast cells. This complex is considered to be composed of the catalytic subunit Chs3 and the regulatory subunit Chs4, both of which are phosphoproteins and transported to the plasma membrane by different trafficking routes. During cytokinesis, Chs3 associates with Chs4 and other proteins at the septin ring, which results in an active CSIII complex. In this study, we focused on the role of Chs4 as a regulatory subunit of the CSIII complex. We analyzed the dynamic localization and interaction of Chs3 and Chs4 during cell division, and found that both proteins transiently co-localize and physically interact only during bud formation and later in a period during septum formation and cytokinesis. To identify unknown binding partners of Chs4, we conducted different screening approaches, which yielded several novel candidates of Chs4-binding proteins including the septin-associated kinase Gin4. Our further studies confirmed this interaction and provided first evidence that Chs4 phosphorylation is partially dependent on Gin4, which is required for proper localization of Chs4 at the bud neck.