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. 2022 Aug 8;11:e77035. doi: 10.7554/eLife.77035

Figure 5. The Vrl1 AnkRD associates with the Vin1 N-terminus through electrostatic interactions.

(A) Schematic of query sequences used to predict the interact between Vrl1 and the Vin1 N-terminus. Modelled regions are shown as completely opaque. (B) ColabFold-predicted interaction between the Vrl1 AnkRD and a minimal fragment of the Vin1 N-terminus (Vin176-95; pTMscore = 0.73). (C) Vrl1 sequence conservation within family Saccharomycetaceae determined by ConSurf and mapped to a surface model that was predicted by ColabFold. Strong sequence conservation can be seen at the predicted Vin176-95 interacting site and near the catalytic D373 residue. (D) Top: Vin176-95 is predicted to associate with Vrl1 through a run of basic residues. Bottom: Acidic and polar residues in the predicted Vin1-associating Vrl1 AnkRD site are among the most conserved within family Saccharomycetaceae. (E) Mutation of acidic and polar residues in the Vrl1 AnkRD reduces recruitment of the Vin1 N-terminus by the Vrl1(1-703)YPE chimera. (F) Quantification of Vin11-116-mScI puncta per cell in E. One-way ANOVA with Tukey’s multiple comparison test; n=3, cells/strain/replicate ≥863; not significant, n.s.=p > 0.05, *=p < 0.05, ***=p < 0.001, ****=p < 0.0001. Scale bars, 2 µm. Error bars report SEM. OE, over-expressed. Nt, N-terminus. WT, wild type. aa, amino acids. YPE, Ypt35(PX)-Envy.

Figure 5—source data 1. Data associated with Figure 5F.

Figure 5.

Figure 5—figure supplement 1. Confidence measures of Vrl1 AnkRD-Vin1 N-terminus binding predictions.

Figure 5—figure supplement 1.

(A) ColabFold-predicted interaction between the Vrl1 AnkRD and a minimal fragment of the Vin1 N-terminus (Vin176-95; pTMscore = 0.73) with pLDDT scores mapped to each residue of Vin1 (left) or Vrl1 (right). In each case, the interacting protein partner is shown in gray. (B) ColabFold-generated predicted alignment error (PAE; Jumper et al., 2021; Mirdita et al., 2022) plot for Vrl1(1-703) and the Vin1 N-terminus (Vin11-116) indicates an intermolecular interaction in the off-diagonal boxes. (C) A plot of predicted local-difference distance test (pLDDT; Jumper et al., 2021; Mirdita et al., 2022) scores for five different models of Vrl1(1-703) and Vin11-116 indicates that the minimal region of Vin1 that was recruited by the Vrl1(1-703)YPE chimera, Vin176-95, is the sole confidently predicted region. aa, amino acids. Nt, N-terminus.
Figure 5—figure supplement 2. Vrl1(1-703)YPE chimeras with AnkRD mutations are stably expressed.

Figure 5—figure supplement 2.

AnkRD-containing Vrl1(1-703)YPE chimeras are stable by western blot when mutations are introduced to sites that are predicted to interact with the Vin1 N-terminus. Pgk1 serves as a loading control. OE, over-expressed. WT, wild type. YPE, Ypt35(PX)-Envy.
Figure 5—figure supplement 2—source data 1. Uncropped blot data associated with Figure 5—figure supplement 2.