Figure 1.
Scanning mutagenesis of the Shr3 membrane domain. (A) Graphical representation of Shr3 topology (upper) and AlphaFold-predicted structural model (lower; Jumper et al., 2021; Varadi et al., 2021). The position of residues resulting in a non-functional protein is indicated. Three-dimensional molecular graphics and analyses were performed with UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax; Pettersen et al., 2021). (B) Serial dilutions of cell suspensions from strain JKY2 (shr3Δ) carrying pRS316 (VC), pPL210 (SHR3), pAR4 (shr3-35), pAR18 (shr3-50), or pPL1349 (shr3-76) spotted on YPD and YPD+MM (upper panels). The plates were incubated at 30°C for 2 d and photographed. Immunoblot analysis of Shr3 proteins in extracts prepared from the strains; the levels of Pgk1 were used as loading controls (lower panels). The blots were developed using α-Shr3 and α-Pgk1 antibodies. The signal intensities of the immunoreactive forms of Shr3 and Pgk1 were quantified, and the Shr3 signals were normalized with respect to Pgk1; the mean values are plotted and error bars show standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F1.

Scanning mutagenesis of the Shr3 membrane domain. (A) Graphical representation of Shr3 topology (upper) and AlphaFold-predicted structural model (lower; Jumper et al., 2021; Varadi et al., 2021). The position of residues resulting in a non-functional protein is indicated. Three-dimensional molecular graphics and analyses were performed with UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax; Pettersen et al., 2021). (B) Serial dilutions of cell suspensions from strain JKY2 (shr3Δ) carrying pRS316 (VC), pPL210 (SHR3), pAR4 (shr3-35), pAR18 (shr3-50), or pPL1349 (shr3-76) spotted on YPD and YPD+MM (upper panels). The plates were incubated at 30°C for 2 d and photographed. Immunoblot analysis of Shr3 proteins in extracts prepared from the strains; the levels of Pgk1 were used as loading controls (lower panels). The blots were developed using α-Shr3 and α-Pgk1 antibodies. The signal intensities of the immunoreactive forms of Shr3 and Pgk1 were quantified, and the Shr3 signals were normalized with respect to Pgk1; the mean values are plotted and error bars show standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F1.

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