Flo1 Protein Research Articles

Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

ABSTRACTWe 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: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-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.

The mannose-specific lectin domains of Flo1p fromSaccharomyces cerevisiaeand Lg-Flo1p fromS. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin–carbohydrate complexes

Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris

Saccharomyces cerevisiae flocculation is governed by FLO genes, encoding Flo proteins (flocculins). Flo proteins are cell wall proteins consisting of three domains, sticking out of the cell wall and interacting with other yeast cells using their N-terminal mannose-binding domain. Until recently, flocculation research was focused on the genetic and cellular level. To extend the knowledge about flocculation to the protein level, we isolated the N-terminal domain of the Flo1p (N-Flo1p) that contains the mannose-binding domain, which is responsible for the strong interaction (flocculation) of S. cerevisiae cells. To obtain a high production yield and a more uniform and lower glycosylation of N-Flo1p, it was cloned in Pichia pastoris. The expression and the purification of N-Flo1p were optimised towards a one-step purification protocol. The activity of the protein, i.e. the binding of the purified protein to mannose using fluorescence spectroscopy, was demonstrated.

ManySaccharomyces cerevisiaeCell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell−cell and cell−surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity

The expression of the Flo11 flocculin in Saccharomyces cerevisiae offers the cell a wide range of phenotypes, depending on the strain and the environmental conditions. The most important are pseudohyphae development, invasive growth and flocculation. The mechanism of cellular adhesion mediated by Flo11p is not well understood. Therefore, the N-terminal domain of Flo11p was purified and studied. Although its amino acid sequence shows less similarity with the other flocculins, Flo11p belongs to the flocculin family. However, the N-terminal domain contains the 'Flo11-domain' (PF10181), but not the mannose-binding PA14 domain, which is present in the other flocculins (Flo1p, Flo5p, Flo9p and Flo10p). Structural and binding properties of the N-terminal domain of Flo11p were studied. It is shown that this domain is O-glycosylated and is structurally composed mainly of β-sheets, which is typical for the members of the flocculin family. Furthermore, fluorescence spectroscopy binding studies revealed that N-Flo11p does not bind mannose, which is in contrast to the other Flo proteins. However, surface plasmon resonance analysis showed that N-Flo11p self-interacts and explains the cell-cell interaction capacity of FLO11-expressing cells.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to D-mannose, α-methyl-D-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.

Flocculation protein structure and cell–cell adhesion mechanism in Saccharomyces cerevisiae

Cell-cell adhesion occurs in a broad spectrum of biological processes, of which yeast flocculation is an area of interest for evolutionary scientists to brewers and winemakers. The flocculation mechanism is based on a lectin-carbohydrate interaction but is not yet fully understood, although the first model dates back to the 1950s. This review will update the current understanding of the complex mechanism behind yeast flocculation. Moreover, modern technologies to measure the forces involved in single carbohydrate-lectin interactions, are discussed. The Flo1 protein has been extensively described as the protein responsible for strong flocculation. Recently, more research has been directed to the detailed analysis of this flocculin. Due to the advances in the field of bioinformatics, more information about Flo1p could be obtained via structurally or functionally related proteins. Here, we review the current knowledge of the Flo1 protein, with a strong emphasis towards its structure.

Deletion of Tandem Repeats Causes Flocculation Phenotype Conversion from Flo1 to NewFlo in Saccharomyces cerevisiae

A gene, FLONS, conferring NewFlo-type flocculation ability in yeast was cloned. The 3,396-bp ORF encoded a peptide of 1,132 amino acids with high identity to Flo1 protein. Aligned with the FLO1 gene, two repeated regions (675 and 540 bp) were lost in the middle of FLONS, revealing that this gene was a derived form of the FLO1 gene. The missing repeated sequence contained three highly homologous repeat units. Although the flocculation phenotype of the transformant YTS-S with the FLONS gene was inhibited by both mannose and glucose, it exhibited some distinguished physiological characteristics from the reported typical NewFlo-type flocculation during detailed investigation. The deletion of repeats was suspected to cause conversion of the flocculation phenotype from Flo1 to NewFlo, suggesting that intragenic tandem repeats generated functional variability in Flo1 protein.

Molecular phylogenetics of ascomycotal adhesins—A novel family of putative cell-surface adhesive proteins in fission yeasts

In this work, we identify a family of putative adhesins in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. The members of this family share a conserved tandem repeat related to those found in the Candida albicans Als family of adhesins. Unlike previously characterised adhesins that possess conserved ligand-binding domains at the N-terminus, this group of proteins carry ligand-binding domains at their C-termini. We demonstrate that one such domain—the uncharacterised GLEYA domain, is related to the lectin-like ligand-binding domain found in the Saccharomyces cerevisiae Flo proteins. Unlike the Flo and Als proteins, the fission yeast adhesins do not contain detectable glycosyl phosphatidyl inositol (GPI) membrane anchor signals to mediate their attachment to the cell wall, which may suggest a novel cell wall attachment mechanism. Further sequence analysis identified several putative adhesins in the sub-phylum of Pezizomycotina, where only a few adhesins have been described to date.

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.

Preparation of Yeast Strains Displaying IgG Binding Domain ZZ and Enhanced Green Fluorescent Protein for Novel Antigen Detection Systems

To develop novel immunofluorescence-labeling and antigen-detection systems, the ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G, and enhanced green fluorescent protein (EGFP) were displayed on the cell surface of Saccharomyces cerevisiae by cell-surface engineering using the C-terminus 318 amino acids of Flo1 protein. Two systems were constructed, one for co-display of ZZ and EGFP, and one for display of a fusion protein of the two. In both cases, two proteins on the cell surface successfully retain their activities.

Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates.

We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.

Region of FLO1 proteins responsible for sugar recognition.

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.

Distribution of the flocculation protein, flop, at the cell surface during yeast growth: the availability of flop determines the flocculation level.

The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation. We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence. Our data show that they are not permanently present at the cell surface but that their amount increases during growth. With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level. Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins. The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes. Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds. Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells. Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins. Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment.

The Saccharomyces cerevisiae FLO1 flocculation gene encodes for a cell surface protein.

The sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined as FLO5 (Bidard et al., 1994) has revealed that it was a FLO1 gene. The FLO1 gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy. The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein. A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level. A gene disruption experiment has shown that FLO5 strain STX 347-1D contains at least two flocculation genes of the FLO1 type but that they are supposed to be inactive and do not contribute to its flocculation. However, enzyme-linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of the FLO5 strain STX 347-1D is antigenically related to Flo1p. A deletion analysis of the 5' region of the FLO1 gene has shown that the expression is submitted to controls which depend on the genetic background of the strain.

Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13, 47-52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47, 2889-2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.

Molecular cloning and analysis of the yeast flocculation gene FLO1.

The DNA sequence of the flocculation gene FLO1 of Saccharomyces cerevisiae, which is located on chromosome I (Watari et al., 1989) was determined. The sequence contains a large open reading frame (ORF) of 2586 bp and codes for a protein of 862 amino acids. However, further study (genomic Southern and polymerase chain reaction analyses) indicated that the gene we cloned was not the intact FLO1 gene but a form with an approximately 2 kb deletion in the ORF region. The intact FLO1 gene was then cloned and its nucleotide sequence determined. The sequence revealed that the ORF of the intact gene is composed of 4611 bp which code for a protein of 1537 amino acids. A remarkable feature of the putative Flo1 protein is that it contains four families of repeated sequences composed of 18, 2, 3 and 3 repeats and that it has a large number of serines and threonines. In the deleted FLO1 form, a large part of these repeated sequences was missing. The N- and C-terminal regions are hydrophobic and both contain a potential membrane-spanning region, suggesting that the Flo1 protein is an integral membrane protein and a cell wall component.