Figure 3.
Mutational analysis of Shr3 function and substrate specificity. (A) Summary of growth characteristics of JKY2 (shr3Δ) individually expressing 44 Shr3-mutant proteins. Cells were spotted on media containing toxic amino analogs as described in Materials and methods. Growth was scored after 2–3 d of incubation at 30°C (Fig. S1). Colors reflect Shr3 function relative to wild-type activity: red, no function (−); orange, weak but detectable function (±); yellow, intermediate function but less than wild-type (+); light blue, wild-type function (WT); and dark blue, enhanced function (WT+). (B) Clustal O (Madeira et al., 2019) comparison of Shr3 sequences, corresponding to aa residues 1–159 of S. cerevisiae, and orthologs of members from the Saccharomyces sensu stricto group (S. paradoxus, S. mikatae) and orthologs from sensu lato fungi (S. pombe, A. nidulans, and C. albicans). The consensus plot (identity; Waterhouse et al., 2009) and detailed multiple sequence alignments are presented for the regions with mutations giving rise to major growth defects on selective media; identical residues in three (light blue), four (blue), and five or six homologs (dark blue) are highlighted. (C) Shr3-dependent Ssy1 folding and function assessed by Stp1 processing. Immunoblot analysis of extracts from FGY135 (shr3Δ) carrying pCA204 (STP1-13xMYC) and pRS316 (VC), pPL210 (SHR3), pAR004 (shr3-35), pAR45 (SHR3Δ94), pAR018 (shr3-50), or pPL1351 (shr3-76). Cells were grown in SD and induced for 30 min with 1.3 mM leucine (+) as indicated. (D) Predicted Shr3 structure (AlphaFold) with residues colored by sequence conservation ranging from cyan for less conserved (conservation equal or lower to a value of −0.5) to maroon for more conserved positions (conservation equal or higher to a value of 2.5). The coloring is assigned via ChimeraX, which uses the multiple sequence alignment PF08229_rp55 from Pfam (https://pfam.xfam.org/family/PF08229#tabview=tab3) and calculates conservation based on the entropy-based measure from AL2CO (Pei and Grishin, 2001). (E) Mutation-induced structural alterations predicted using AlphaFold. The individual models (yellow) of shr3-35 (left), shr3Δ92 (center), and Shr3Δ94 (right; rotated for better visualization of predicted changes) are superimposed onto the wild-type Shr3 structure (light blue). Molecular graphics and analyses were performed with UCSF ChimeraX. Source data are available for this figure: SourceData F3.

Mutational analysis of Shr3 function and substrate specificity. (A) Summary of growth characteristics of JKY2 (shr3Δ) individually expressing 44 Shr3-mutant proteins. Cells were spotted on media containing toxic amino analogs as described in Materials and methods. Growth was scored after 2–3 d of incubation at 30°C (Fig. S1). Colors reflect Shr3 function relative to wild-type activity: red, no function (−); orange, weak but detectable function (±); yellow, intermediate function but less than wild-type (+); light blue, wild-type function (WT); and dark blue, enhanced function (WT+). (B) Clustal O (Madeira et al., 2019) comparison of Shr3 sequences, corresponding to aa residues 1–159 of S. cerevisiae, and orthologs of members from the Saccharomyces sensu stricto group (S. paradoxus, S. mikatae) and orthologs from sensu lato fungi (S. pombe, A. nidulans, and C. albicans). The consensus plot (identity; Waterhouse et al., 2009) and detailed multiple sequence alignments are presented for the regions with mutations giving rise to major growth defects on selective media; identical residues in three (light blue), four (blue), and five or six homologs (dark blue) are highlighted. (C) Shr3-dependent Ssy1 folding and function assessed by Stp1 processing. Immunoblot analysis of extracts from FGY135 (shr3Δ) carrying pCA204 (STP1-13xMYC) and pRS316 (VC), pPL210 (SHR3), pAR004 (shr3-35), pAR45 (SHR3Δ94), pAR018 (shr3-50), or pPL1351 (shr3-76). Cells were grown in SD and induced for 30 min with 1.3 mM leucine (+) as indicated. (D) Predicted Shr3 structure (AlphaFold) with residues colored by sequence conservation ranging from cyan for less conserved (conservation equal or lower to a value of −0.5) to maroon for more conserved positions (conservation equal or higher to a value of 2.5). The coloring is assigned via ChimeraX, which uses the multiple sequence alignment PF08229_rp55 from Pfam (https://pfam.xfam.org/family/PF08229#tabview=tab3) and calculates conservation based on the entropy-based measure from AL2CO (Pei and Grishin, 2001). (E) Mutation-induced structural alterations predicted using AlphaFold. The individual models (yellow) of shr3-35 (left), shr3Δ92 (center), and Shr3Δ94 (right; rotated for better visualization of predicted changes) are superimposed onto the wild-type Shr3 structure (light blue). Molecular graphics and analyses were performed with UCSF ChimeraX. Source data are available for this figure: SourceData F3.

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