The dual role of amyloid-β-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae
- Toulouse Biotechnology Institute, INSA, France
- Lallemand, Lallemand SAS, France
- LAAS, CNRS, France
Figures
Figure 1 with 1 supplement
Cell surface analysis of the L69 strain using silicon nitride (Si3N4) atomic force microscopy (AFM) cantilevers.
AFM height images (A) of a single yeast cell from L69 strain embedded in polydimethylsiloxane (PDMS) microtimber are shown. In (B) an AFM contact image is shown in which the topography region that …
Figure 1—figure supplement 1
Cell surface analysis of BY4741 and L69 strains using silicon nitride (Si3N4) atomic force microscopy (AFM) cantilevers.
A single cell from strain BY4741 and L69 was trapped in the polydimethylsiloxane (PDMS) chamber as described in Materials and methods. AFM height image (z-scale: 2 µm) (A) and adhesion image …
Figure 2 with 1 supplement
The nanosized patches at the cell surface of L69 strain are FLO11-dependent adhesion nanodomains that are abolished by anti-amyloïd compounds.
In (A) is shown the atomic force microscopy (AFM) adhesion image of a cell from L69 strain before and 90 min after treatment with 5 µM of the anti-amyloid peptide VASTTV. In (B) is shown the …
Figure 2—figure supplement 1
Expression levels of FLO11 in L69 and BY4741 strain measured by quantitative reverse transcription PCR (qRT-PCR).
Transcript levels of FLO11 were determined in exponential cultures of L69 and BY4741 strains on YPD (A) or on YNGal supplemented with required amino acids, uracil, and adenine at 0.1% (w/v), except …
Figure 3 with 5 supplements
Hydrophobic cluster analysis (HCA) of the Flo11 protein from various Saccharomyces cerevisiae strain, and quantitative reverse transcription PCR (qRT-PCR) of wild-type and mutated FLO11 genes.
In (A) is reported the HCA plots of Flo11p from wine yeast strain L69, Flor strain 133d, and laboratory strains BY4741 and Σ1278b. The three domains of the proteins as determined using various …
Figure 3—figure supplement 1
Sequences alignment of Flo11p from L69, BY4741, S1278b, and 133d strains.
The Flo11p sequences were aligned with Clustal Omega. The sequence of Flo11p from L69 strain was retrieved from the genome sequence of this strain (Lallemand Inc, unpublished data), whereas Flo11p …
Figure 3—figure supplement 2
Schematic representation of the Flo11 protein sequence from BY4741 strain by hydrophobic cluster analysis.
The Flo11p from BY4741 strain is shown with the N-terminal secretion signal sequences boxed in blue determined with SignalP-4.1 server and the C-terminal glycosylphosphatidylinositol (GPI) addition …
Figure 3—figure supplement 3
Schematic representation of the Flo11 protein sequence from S1278b strain by hydrophobic cluster analysis.
Same description as in Figure 4.
Figure 3—figure supplement 4
Schematic representation of the Flo11 protein sequence from L69 strain by hydrophobic cluster analysis (HCA).
Same as in Figure 4. Notice the presence of two additional amyloid-forming sequences, together with two additional β-aggregation motifs ITTTFV in this protein that are not present in Flo11p of all …
Figure 3—figure supplement 5
Schematic representation of the Flo11 protein sequence from flor yeast strain 133d by hydrophobic cluster analysis.
Same description as in Figure 4.
Figure 4
The cell wall localization of Flo11p is not impaired by removal of C-terminus, as well as of N-terminus or ablation of RR2 region bearing amyloid-core sequences.
Localization of Flo11p and its variants was visualized by confocal microscopy in strain L69 that expressed a 6x-His-tag at the C-terminus of the Flo11 or its variants from the endogenous FLO11 …
Figure 5 with 3 supplements
Domain in the Flo11p of L69 responsible for the production of adhesive nanodomains.
In panel (A) are shown atomic force microscopy (AFM) height image (a) and adhesion image (b) of a single cell from L69 strain expressing Flo11p defective of the N-terminus (flo11-ΔNter), C-terminus …
Figure 5—figure supplement 1
Cell surface analysis of the L69flo11-ΔNter strain using silicon nitride (Si3N4) atomic force microscopy (AFM) cantilevers.
AFM height image (A, B), adhesion image from the hatched square in A (C) and stiffness image (E) of a single yeast cell from L69 flo11-DNter trapped in a polydimethylsiloxane (PDMS) chamber is …
Figure 5—figure supplement 2
Cell surface analysis of the L69flo11-ΔCter strain using silicon nitride (Si3N4) atomic force microscopy (AFM) cantilevers.
AFM height image (A, B), adhesion image (C), and stiffness image (D) of a single yeast cell from L69 flo11-DCter is shown. In (E) is shown the height of the patches versus their size as indicated by …
Figure 5—figure supplement 3
Cell surface analysis of L69flo11-ΔRR1 using silicon nitride (Si3N4) atomic force microscopy (AFM) cantilevers.
Representative force-distance curves (right) at different location on the AFM adhesion image (left) indicated by a number obtained from a single cells of L69flo11-DRR1 embedded in …
Figure 6 with 1 supplement
The Flo11p-dependent cell-cell aggregation is potentiated by amyloid-forming sequences.
The yeast cells were cultivated as in Figure 5 but until entry in stationary phase and observed under an optical microscope. In (A) are shown representative photographs of cell aggregates from the …
Figure 6—figure supplement 1
Effect of thioflavine S on the Flo11p-dependent cell-cell aggregation.
BY4741 transformed with pYES2.1 carrying either FLO11BY or FLO11L69, L69, L69flo11-∆RR2 (expressing Flo11p variant lacking RR2), and YSWT3a (haploid strain derived from S1278b) were cultivated in …
Figure 7
Adherence to plastic is strongly impacted by pH and does not solely depend on the A-domain of Flo11p.
Adherence of yeast cells was carried out a 96-well polystyrene plate as described in Materials and methods. The data are the mean of three replicates measurements ± standard deviation. Significant …
Figure 8 with 1 supplement
Surface hydrophobicity mainly depends on abundance of Flo11p.
Surface hydrophobicity corresponded to the percentage of cells partitioning in the octane layer, as described in Materials and methods. The data are the mean of three biological replicates and …
Figure 8—figure supplement 1
Assay of velum formation.
The assay was carried out by static incubation for 7 days of yeast cells in the flor medium at 23°C.
Figure 9 with 2 supplements
The Invasive growth in agar is abolished by deletion of either N- or C-terminal of the Flo11p.
All strains were pre-grown in YNGal that was supplemented with the auxotrophic requirements when needed (i.e. uracil, leucine, histidine, methionine at 0.1% for BY4741, BYflo11Δ, and YSTW3α but only …
Figure 9—figure supplement 1
Invasive growth in agar by different yeast strains, and impact of the culture medium and of the domains of FLO11p on this phenotype.
Yeast cells were initially cultivated in YNGal complemented with amino acids and uracil, except for BYflo11D and Sflo11D expressing FLO11BY or FLO11L69 on a pYES2.1 plasmid for which uracil was …
Figure 9—figure supplement 2
Sensitivity of L69 and L69 mutant strains to caffeine and calcofluor white (CFW) drugs.
Cells were exponentially grown in YPD medium and were collected by centrifugation, then resuspended in sterilized water at 108 cells/ml; 5 µl of this cell suspension diluted 10–105 fold was spotted …
Tables
Table 1
β-Aggregation-prone sequences in Flo11 of various Saccharomyces cerevisiae strains*.
| Amyloid sequence | Amino acid position | % β-Aggregation | |
|---|---|---|---|
| Flo11pL69 | VVSTTVVVTTAVT | 1178; 1285; 13921494 | 75.759.4 |
| Flo11pBY | VVSTTVVVTTAVT | 10331494 | 75.884.8 |
| Flo11pΣ | VVSTTVVVTTAVT | 881983 | 75.659 |
| Flo11p133d | VVSTTVVVTTAVT | 13011403 | 70.959.1 |
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*
TANGO software (http://tango.crg.es/) with default settings for pH, ionic strength, and temperature was used to determine Flo11p regions with β-aggregation potential superior to 30%.
Additional files
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Supplementary file 1
TANGO software analysis of β-aggregation motifs in Flo1, Flo5, Flo9, Flo10, and Flo11 protein from Saccharomyces cerevisiae S288c strain.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp1-v2.docx
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Supplementary file 2
Identification of sequence repeats and beta-aggregation prone sequence in Flo11 proteins from different yeast strains.
(a) Search for intragenic repeats using EMBOSS ETANDEM software. (b) Search for β-aggregation-prone sequence in the different Flo11 proteins using TANGO software (http://tango.crg.es/). *β-Aggregation-prone sequences > 30% were searched using TANGO software (at http://tango.crg.es/) with default setting of pH, ionic strength, and temperature. Amyloid-core sequences are highlighted in yellow.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp2-v2.docx
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Supplementary file 3
Proteomic analysis of cell wall/cell membrane preparation from L69 strain.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp3-v2.xlsx
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Supplementary file 4
Yeast strains used or constructed in this study.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp4-v2.docx
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Supplementary file 5
Plasmids constructed in this work.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp5-v2.docx
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Supplementary file 6
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/68592/elife-68592-supp6-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68592/elife-68592-transrepform1-v2.docx
