Journal: Journal of Fungi

Article Title: Structure of the Yeast Cell Wall Integrity Sensor Wsc1 Reveals an Essential Role of Surface-Exposed Aromatic Clusters

doi: 10.3390/jof8040379

Figure Lengend Snippet: Functional analysis of surface-exposed aromatic residues in the CRD of Sc Wsc1. ( a ) A yeast strain carrying a chromosomal deletion of the WSC1 gene ( wsc1∆ ; YHUM2959) was transformed with an empty control plasmid (control; pRS314) or plasmids carrying either WT WSC1 (BHUM3303) or WSC1 lacking the CRD ( ∆CRD ; BHUM3308) or WSC1 with mutations in one of the aromatic clusters 1 (Y22A Y24A Y107A; BHUM3304), 2 (Y64A Y70A, Y104A; BHUM3305), or 3 (Y41A W43A Y89A F91A Y93A; BHUM3306). All WSC1 variants on the plasmids carry a C-terminal translational fusion to the green fluorescent protein mNeonGreen. As an additional control, a yeast strain carrying an untagged chromosomal WSC1 gene ( WSC1 ; ESM356-1) and an empty control plasmid (pRS314) was used (shown on the left). Plasmid carrying strains were grown to logarithmic phase in liquid SC-Trp medium, and fivefold serial dilutions of the cultures were spotted onto solid YPD medium or YPD medium supplemented with either 0.4 µg/mL caspofungin (Caspofungin), 30 µg/mL Congo red (Congo Red), 100 µg/mL Calcofluor white (Calcofluor), or 2 mg/mL caffeine (Caffeine). Plates were photographed after incubation for 3–5 days at 30 °C. The pictures shown are representative for at least two independent transformants. ( b ) Western blot analysis. Strains described in ( a ) were grown to logarithmic phase, and full-length Wsc1-mNeonGreen proteins were detected in cell extracts by anti-mNeonGreen antibodies (α-NG). As an internal loading control, tubulin was detected using anti-tubulin antibodies (α-Tub). The pictures shown are representative for at least two independent transformants. ( c ) Microscopy. All strains were further analyzed for expression of WSC1 variants by fluorescence microscopy after growth to logarithmic phase using the channels for transmission light (TL) or for mNeonGreen (GFP). The white bar corresponds to 5 µm.

Article Snippet: Yeast strains expressing different mNeonGreen -tagged WSC1 gene variants were analyzed by microscopy after growth to logarithmic phase under a Zeiss Axiovert 200M microscope using (i) transmission light microscopy (TM) and (ii) fluorescence microscopy with a GFP filter set (AHF Analysentechnik AG, Tübingen, Germany).

Techniques: Functional Assay, Transformation Assay, Control, Plasmid Preparation, Incubation, Western Blot, Microscopy, Expressing, Fluorescence, Transmission Assay