Functional analyses in yeast loss-of-function mutations in conserved residues of BtpB.

(A) Alignment of protein sequences of the TIR domains of BtpB, BtpA, human SARM1 and plant RUN1. Conserved residues relevant for this study are marked with the same color code as in Fig 4, except for for the catalytic site residues W213 and E217, that are colored in pink. (B) Structure of BtpA-TIR (left; PDB: 4LZP)) and RUN1-NADP+ complex (right; PDB: 6O0W), showing the equivalent positions of residues mutated in BtpB isolated in the yeast screen. Both structures cartoons are displayed in the same orientation. Side chain of mutated residues of BtpA relevant for this study are colored as in (A). The side chains of residues of the catalytic site of RUN1 are shown as ball-and-sticks and colored in pink and the NADP+ ligand is colored in cyan. Specific atoms are colored as follows: nitrogen in blue, oxygen in red and phosphorus in orange. (C) Phenotype of selected loss-of-function BtpB mutants. Ten-fold serial dilution growth assay of YPH499 cells transformed with pYES2 empty vector and pYES2 plasmid derivatives expressing full-length BtpB wild-type and mutants D158G, S162P and Y225C, under control (Glucose) and induction (Galactose) conditions. (D) Nomarski and fluorescence microscopy images of YPH499 cells expressing the GFP-BtpB indicated mutants, after 4h induction, stained with the endocytic marker FM4-64 for 1h. Scale bars indicate 5 μm. (E) Graph from the same experiment as in C representing the percentage of cells showing both GFP and FM4-64 vacuolar signal. Results correspond to means ± standard deviation of three independent transformants (n ≥ 100) and statistical comparison was done with one-way ANOVA with a p-value < 0.0001 (***) for all four mutants versus wild-type. (PDF)