Supplementary materials for:
“Evolutionary reshaping of fungal mating pathway scaffold proteins”.
Pierre Côte‡, Traian Sulea, Daniel Dignard, Cunle Wu and Malcolm Whiteway‡*.
Genetics Group, Biotechnology Research Institute,
National Research Council of Canada,
6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2
‡ Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1
* Corresponding author:
Dr. Malcolm Whiteway
Genetics Group,
Biotechnology Research Institute,
National Research Council of Canada,
6100 Royalmount Ave., Montreal, Quebec,
Canada H4P 2R2
Tel: 001-514-496-6146
Fax: 001-514-496-6213
Email:
Supplementary materials
Experimental procedures
Comparative sequence analysis, domain annotation, and structural homology modeling
Homologous protein sequences were retrieved by either BLASTP or TBLASTN search (1) in the Fungal genome database ( and by protein domain architecture search in the SMART database ( 2). Comparative sequence analysis of the assembled datasets (29 Far1-like, 19 Ste5-like and 31 Ste11-like sequences) was carried out with the MAFFT 6 program (3) for deriving multiple sequence alignments with the L-INS-i iterative refinement algorithm and to generate phylogenetic trees, and visualized using Jalview 2.4.0B2 (4). Complete set of all sequences, as well as their accession number, is available at
Structural domain detection for representative Far1-like and Ste5-like proteins was carried out at the Structure Prediction Meta Server ( which assembles state-of-the-art fold recognition methods, and provides a consensus sequence-to-structure scoring using the 3D-Jury meta-predictor (5). In order to confirm the available domain annotations (e.g., from InterPro and member databases, as well as previous reports of predicted domains in some family members (6, 7, 8), full-length sequences were queried. Iteratively, reliably detected domains were excised out and the flanking sequences were subjected to new rounds of fold detection. Finally, the excised sequences of detected domains were re-submitted to the Structure Prediction Meta Server to obtain the final template ranking, reliability indicators and query-to-template sequence alignments. Multiple-queries-multiple-templates sequence alignments were assembled using the (i) MAFFT multiple sequence alignment of the queries, (ii) 3D-Coffee structure-based sequence alignment of the templates (9), (iii) 3D-Jury consensus fold recognition based alignment between queries and templates, (iv) manual minor local improvements in the overall secondary structure alignment, and (v) 3D-structural alignment using the domain alternate fit in Swiss-PdbViewer v4.0.1 (10). Secondary structure predictions were based on three methods: PROFsec (11), PSI-PRED (12) and Jnet (13), from which a consensus was derived for each sequence by majority voting.
Potential nuclear localization signals/plasma membrane-binding (NLS/PM) basic motifs and kinase docking sites were inferred from the multiple sequence alignment at positions corresponding to the respective motifs confirmed in S. cerevisiae (14, 15, 16, 17). No attempt was made here to locate such motifs elsewhere in the homologous sequences.
Homology modeling of the C. albicans Ste11 kinase domain was done with the MODELLER 9v1 program (18). Suitable template structures were located by BLASTP searches ( against sequences in the PDB database ( using the C. albicans Ste11 kinase domain and activation loop sequences as queries. The structure of activated human PAK5 phosphorylated at Ser602 and bound to an ATP-competitive inhibitor (PDB code 2F57) was used as template (19). This template structure provides a good overall alignment of the kinase domain and also has suitable homology for constructing the activation loop comprising the acidic insert Asp705-Asp710 probed in this study. The model of C. albicans Ste11 kinase domain phosphorylated at Ser719 was refined by energy minimization using the AMBER force-field (20). An unphosphorylated model was also obtained in which the conformation of the Ser702-Thr714 sequence in the activation loop was refined in MODELLER for 1000 loop generation cycles.
Mapping of protein-protein interactions among Cst5 and other components of the mating pheromone pathway
To map protein-protein interactions among domains of the pheromone signaling pathway components, we used an alternative yeast two-hybrid system (Y2H) we developed recently for the detection of protein-protein interactions in the cytoplasm (detailed information of this Y2H system will be published elsewhere). Briefly, this yeast two-hybrid system is based on the interaction of Ste11p (MEKK) and Ste50p that is required for the HOG pathway activation and osmoadaptation, which is critical for the survival of yeast cells under hyperosmotic stress in the absence the two-component osmosensing branch (Figure 3A). The interaction of Ste11p and Ste50p through their respective SAM domains that is required to activate the HOG pathway can be replaced by interaction of other protein interacting modules (21). This property offers a unique potential to analyze bait-prey interactions by substituting them for the respective SAM domains and using the activation of the HOG pathway as a reporter (Figure 3B and 3C).
Plasmid pYL40 contains the fragment of STE11 encoding Ste11p lacking its SAM domain (aa 110-717) at the SalI siteof pGREG506 (22), and a HIS3 stuffer marker inserted at the SmaI site in front the Ste11SAM. Similarly, pYL45 contains the fragment of STE50 encoding Ste50p without its SAM domain (aa 115-346) at the SalI siteof pGREG503 (22), and a URA3 stuffer marker inserted at the SmaI site in front the Ste50SAM.
All candidate ORFs or their fragments were PCR amplified and cloned into the vector plasmids pYL40 and pYL45 at the SmaI site through in vivo recombination (IVR) in yeast strains YCW1476 and YCW1477. All the primers used for the PCR amplification reactions contain gene specific sequences and common sequences used for IVR in a layout as follows: 5’-ATTCTAGAGCGGCCGCACTAGTGGATCCCCCGGG-gene specific sequence (starting with ATG)-3’ for the forward orientation, and 5’-TCGATAAGCTTGATATCGAATTCCTGCAGCCCGGG-gene specific sequence (delete the stop codon)-3’ for the reverse orientation. To query the bait-prey interaction, IVR positive clones (stuffer marker negative with right inserts) of the baits in one of the two yeast strains were crossed to the IVR positive clones of the preys in the other yeast strain, mating products were selected and their ability to activate the HOG pathway measured by the ability to grow on hyperosmolarity media (Figure 3D&E).
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