Stefan Brückner

Choosing the right lifestyle: adhesion and development in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a eukaryotic microorganism that is able to choose between different unicellular and multicellular lifestyles. The potential of individual yeast cells to switch between different growth modes is advantageous for optimal dissemination, protection and substrate colonization at the population level. A crucial step in lifestyle adaptation is the control of self- and foreign adhesion. For this purpose, S. cerevisiae contains a set of cell wall-associated proteins, which confer adhesion to diverse biotic and abiotic surfaces. Here, we provide an overview of different aspects of S. cerevisiae adhesion, including a detailed description of known lifestyles, recent insights into adhesin structure and function and an outline of the complex regulatory network for adhesin gene regulation. Our review shows that S. cerevisiae is a model system suitable for studying not only the mechanisms and regulation of cell adhesion, but also the role of this process in microbial development, ecology and evolution.

Structural Basis of Flocculin-Mediated Social Behavior in Yeast

In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of “greenbeard” genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 Å. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self- instead of unsocial self-interactions are favored.

Differential regulation of Tec1 by Fus3 and Kss1 confers signaling specificity in yeast development

Transcriptional regulation by mitogen-activated protein (MAP) kinase signaling cascades is a major control mechanism for eukaryotic development. In budding yeast, Fus3 and Kss1 are two MAP kinases that control two distinct developmental programs—mating and invasive growth. We investigated whether signal-specific activation of mating and invasive growth involves regulation of the transcription factor Tec1 by Fus3 and Kss1. We present evidence that, during mating, Fus3 phosphorylates Tec1 to downregulate this invasive growth-specific transcription factor and its target genes. This function of Fus3 is essential for correct execution of the mating program and is not shared by Kss1. We find that Kss1 controls the activity of Tec1 mainly during invasive growth by control of TEC1 gene expression. Our study suggests that signaling specificity can arise from differential regulation of a single transcription factor by two MAP kinases with shared functions in distinct developmental programs.

Structural Basis for Promiscuity and Specificity During Candida Glabrata Invasion of Host Epithelia.

The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization.

The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms

ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

Interactions by the Fungal Flo11 Adhesin Depend on a Fibronectin Type III-Like Adhesin Domain Girdled by Aromatic Bands.

Saccharomyces cerevisiae harbors a family of GPI-anchored cell wall proteins for interaction with its environment. The flocculin Flo11, a major representative of these fungal adhesins, confers formation of different types of multicellular structures such as biofilms, flors, or filaments. To understand these environment-dependent growth phenotypes on a molecular level, we solved the crystal structure of the N-terminal Flo11A domain at 0.89-Å resolution. Besides a hydrophobic apical region, the Flo11A domain consists of a β sandwich of the fibronectin type III domain (FN3). We further show that homophilic Flo11-Flo11 interactions and heterophilic Flo11-plastic interactions solely depend on the Flo11A domain and are strongly pH dependent. These functions of Flo11A involve an apical region with its surface-exposed aromatic band, which is accompanied by acidic stretches. Together with electron microscopic reconstructions of yeast cell-cell contact sites, our data suggest that Flo11 acts as a spacer-like, pH-sensitive adhesin that resembles a membrane-tethered hydrophobin.

The TEA Transcription Factor Tec1 Links TOR and MAPK Pathways to Coordinate Yeast Development

In Saccharomyces cerevisiae, the TEA transcription factor Tec1 controls several developmental programs in response to nutrients and pheromones. Tec1 is targeted by the pheromone-responsive Fus3/Kss1 mitogen-activated protein kinase (MAPK) cascade, which destabilizes the transcription factor to ensure efficient mating of sexual partner cells. The regulation of Tec1 by signaling pathways that control cell division and development in response to nutrients, however, is not known. Here, we show that Tec1 protein stability is under control of the nutrient-sensitive target of rapamycin complex 1 (TORC1) signaling pathway via the Tip41-Tap42-Sit4 branch. We further show that degradation of Tec1 upon inhibition of TORC1 by rapamycin does not involve polyubiquitylation and appears to be proteasome independent. However, rapamycin-induced Tec1 degradation depends on the HECT ubiquitin ligase Rsp5, which physically interacts with Tec1 via conserved PxY motives. We further demonstrate that rapamycin and mating pheromone control Tec1 protein stability through distinct mechanisms by targeting different domains of the transcription factor. Finally, we show that Tec1 is a positive regulator of yeast chronological lifespan (CLS), a known TORC1-regulated process. Our findings indicate that in yeast, Tec1 links TORC1 and MAPK signaling pathways to coordinate control of cellular development in response to different stimuli.

The Transcription Factors Tec1 and Ste12 Interact with Coregulators Msa1 and Msa2 To Activate Adhesion and Multicellular Development

ABSTRACT In Saccharomyces cerevisiae and related yeast species, the TEA transcription factor Tec1, together with a second transcription factor, Ste12, controls development, including cell adhesion and filament formation. Tec1-Ste12 complexes control target genes through Tec1 binding sites (TEA consensus sequences [TCSs]) that can be further combined with Ste12 binding sites (pheromone response elements [PREs]) for cooperative DNA binding. The activity of Tec1-Ste12 complexes is known to be under negative control of the Dig1 and Dig2 (Dig1/2) transcriptional corepressors that confer regulation by upstream signaling pathways. Here, we found that Tec1 and Ste12 can associate with the transcriptional coregulators Msa1 and Msa2 (Msa1/2), which were previously found to associate with the cell cycle transcription factor complexes SBF (Swi4/Swi6 cell cycle box binding factor) and MBF (Mbp1/Swi6 cell cycle box binding factor) to control G1-specific transcription. We further show that Tec1-Ste12-Msa1/2 complexes (i) do not contain Swi4 or Mbp1, (ii) assemble at single TCSs or combined TCS-PREs in vitro, and (iii) coregulate genes involved in adhesive and filamentous growth by direct promoter binding in vivo. Finally, we found that, in contrast to Dig proteins, Msa1/2 seem to act as coactivators that enhance the transcriptional activity of Tec1-Ste12. Taken together, our findings add an additional layer of complexity to our understanding of the control mechanisms exerted by the evolutionarily conserved TEA domain and Ste12-like transcription factors.