title: Molecular mechanism of flocculation self-recognition in yeast and its role in mating and survival.
abstract: We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca(2+) takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca(2+). These results unify the generally accepted lectin hypothesis with the historically first-proposed "Ca(2+)-bridge" hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating.
  
  
  OBJECTIVE
  Yeast cells can form multicellular clumps under adverse growth conditions that protect cells from harsh environmental stresses. The floc formation is based on the self-interaction of Flo proteins via an N-terminal PA14 lectin domain. We have focused on the flocculation mechanism and its role. We found that carbohydrate specificity and affinity are determined by the accessibility of the binding site of the Flo proteins where the external loops in the ligand-binding domains are involved in glycan recognition specificity. We demonstrated that, in addition to the Flo lectin-glycan interaction, glycan-glycan interactions also contribute significantly to cell-cell recognition and interaction. Additionally, we show that flocculation provides a uniquely organized multicellular ultrastructure that is suitable to induce and accomplish cell mating. Therefore, flocculation is an important mechanism to enhance long-term yeast survival.
authors: Department of Bioengineering Sciences, Structural Biology Research Center, Vrije Universiteit Brussel, Brussels, Belgium.
authors: Katty V Y
authors: KV
authors: Goossens
authors: Department of Bioengineering Sciences, Structural Biology Research Center, Vrije Universiteit Brussel, Brussels, Belgium.
authors: Francesco S
authors: FS
authors: Ielasi
authors: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Intawat
authors: I
authors: Nookaew
authors: Ingeborg
authors: I
authors: Stals
authors: Laboratoire de Physique de la Matière Vivante, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
authors: Livan
authors: L
authors: Alonso-Sarduy
authors: Centre for Malting and Brewing Sciences, KU Leuven, Heverlee, Belgium.
authors: Luk
authors: L
authors: Daenen
authors: Centre for Malting and Brewing Sciences, KU Leuven, Heverlee, Belgium.
authors: Sebastiaan E
authors: SE
authors: Van Mulders
authors: Department Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium.
authors: Catherine
authors: C
authors: Stassen
authors: VIB Nucleomics Core, VIB, Leuven, Belgium.
authors: Rudy G E
authors: RG
authors: van Eijsden
authors: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Verena
authors: V
authors: Siewers
authors: Centre for Malting and Brewing Sciences, KU Leuven, Heverlee, Belgium.
authors: Freddy R
authors: FR
authors: Delvaux
authors: Laboratoire de Physique de la Matière Vivante, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
authors: Sandor
authors: S
authors: Kasas
authors: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Jens
authors: J
authors: Nielsen
authors: Department Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium.
authors: Bart
authors: B
authors: Devreese
authors: Department of Bioengineering Sciences, Structural Biology Research Center, Vrije Universiteit Brussel, Brussels, Belgium Ronnie.Willaert@vub.ac.be.
authors: Ronnie G
authors: RG
authors: Willaert
mesh_terms: D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002448:Cell Adhesion; D003227:Conjugation, Genetic; D005427:Flocculation; D020869:Gene Expression Profiling; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000378:metabolism*; D050296:Microbial Viability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D002413:Cations, Divalent; C087578:FLO1 protein, S cerevisiae; D037241:Mannose-Binding Lectins; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium