Fig. 6: 14-3-3 proteins binding to Rim4 RRM3 serve as roadblocks for Rim4 foci assembly.

A Top: schematic of Rim4 with Bmh1/2 binding sites (BBSs). BBS₁: QYTRRYT*YPIS; BBS₂: ELNRRFS*T*HGKI; BBS₃: MPLRRKS*MPNS; BBS₄: RYARRSS*YGYN. Underlined residues: Rabbit-anti-RRXS/T-p antibody recognized sequences. *: key phosphorylation sites responsible for the antibody recognition. Bottom: Representative immunoblotting (IB) from 3 independent repeats with similar results, of the whole-cell lysate (total) and centrifugal fractions. HD, 5% 1,6-Hexanediol (HD) treatment before heat shock. P₁: pellet from 1000 × g centrifugation; P₂₀ and S₂₀: pellet and supernatant from 20,000 × g centrifugation, respectively. Ponceau S staining shows equal loading. B, C Thermal foci formation in cells expressing GFP-Rim4 variants. B Top, mutation sites of Rim4 variants. Bottom, Representative FM images. C GFP-Rim4 variants foci sizes plotted over time. N (foci) = 100 (except for 0 min, when only a few foci were found in each cell. For 0 min, N [foci] = [WT] 9, [BBS_1,2,3,4-C] 15, [BBS_1,3,4-C] 11, [BBS₂-C] 5). Data are shown as mean ± SE. Two-sided Mann–Whiney test for (red) BBS_1,2,3,4-C, (yellow) BBS_1,3,4-C, or (blue) BBS₂-C vs. (black) WT. Statistic marks were color-coded as in the line chart. p-values: a: 0.4608 (ns); b: 0.7632 (ns); c: 0.3116 (ns); d: 1.67e-40 (***); e: 2.41e-26 (***); f: 0.7915 (ns); g: 0.0004 (***); h: 0.0100 (**); i: 0.4917 (ns). D The yeast 14-3-3 proteins Bmh1/2 serve as roadblocks by binding to phosphorylated BBS₂ in RRM₃, preventing Rim4 self-assembly. Heat triggers Rim4 dephosphorylation, resulting in dissociation of Bmh1/2 and hence Rim4 self-assembly (Seed). Scale bars are 5 µm.