Fig. 5: Functional properties of IST2 linker constructs probed in yeast complementation assays.
From: Structural basis for lipid transport at membrane contact sites by the IST2–OSH6 complex
Cell growth assayed upon overexpression of IST2 constructs in a S. cerevisiae ist2;psd1-KO strain. a, Comparison of WT (IST2) to the constructs IST2921 lacking 25 residues of the C terminus including the PM-binding region and IST2716 lacking 230 residues including the OSH6-binding site (P < 0.0001 for both). b, Comparison of WT to a protein carrying the R750A substitution in the OSH6-binding site of IST2 (P = 0.001), which strongly reduced the interaction, and another construct where the sequence of the binding site was scrambled without changing its composition (P < 0.001). c, Comparison of WT to constructs where the OSH6-binding site was moved by 83 residues toward the N or C terminus of the protein (P < 0.0001 for both). d, Comparison of WT to constructs where the IST2 linker was symmetrically shortened by removal of residues from the center of both sequence regions located N-terminally and C-terminally to the binding site to reach the indicated fraction of its original length (IST2 versus neg., P < 0.0001; IST2 versus 75%, P = 0.9994; IST2 versus 50%, P < 0.0001; IST2 versus 33%, P < 0.0001; IST2 versus 25%, P < 0.0001). NS, not significant. e, Comparison of WT to constructs with the OSH6-binding site removed and OSH6 fused into the equivalent position into the linker (fusion, P = 0.0137), a fusion construct where the linker was symmetrically truncated by 50% in the center of both regions flanking the fused LTP (fusion trunc., P < 0.0001) or expanded by 50% by introducing a G-S peptide sequence (Fusion ext., P = 0.9951). In a–e, schemes illustrate the properties of IST2 constructs. Bars display the mean normalized growth compared to WT (n = 3 × 3 replicates for most constructs and 2 × 3 replicates for fusion trunc. and fusion ext.). Errors are the s.e.m. Values of individual experiments are shown as dots. Statistical analysis was performed using a one-way ANOVA and Tukey’s multiple comparisons test. Values from the same experimental replicate were matched in the analysis. f, Potential energy of the C-terminal region of IST2 modeled as an entropic spring during the shuttle of OSH6 between the ER and the PM in constructs of different length. Top, correspondence of energies to states during transport.
