Figure 4. The W-Peptide Is a Localization Signal for the EvC Zone.

(A) Evc2ΔW-YFP (green) is localized along the entire length of cilia (red) in Evc2ΔW cells regardless of Hh signaling (2 hr).

(B) STED image of Evc2ΔW-YFP distribution at cilia.

(C) Evc2ΔW-YFP localization relative to various ciliary compartments (compare with Figure 1H).

(D) Mean (± SD) SAG-induced Hh reporter activity in cells transfected with the indicated deletion mutants of Evc2.

(E) Protein sequence of the region between residues 1177 and 1198 of Evc2 that was analyzed by alanine-scanning mutagenesis. For each of the ten mutants (Evc2A1–A10), the indicated stretch of four aa was mutated to alanines (A). The FV motif, which is critical for suppression of Hh signaling, is circled in red.

(F) Effects of the Evc2 mutants from (E) on Hh signaling induced by SAG (mean ± SD). The red line marks a 50% reduction in reporter activity compared with WT Evc2.

(G) T-Coffee multiple sequence alignment of the W-peptide in mouse, human, chicken, and fugu Evc2. Boxes encircle positions where Ala mutations produce (red) or fail to produce (green) a dominant-negative effect (see Figures S4D and S4E). The red line underlines the region subjected to Ala-scanning mutagenesis in (E). Identical (*), conserved (:) and semiconserved (●) residues are indicated.

(H) YFP-SmoW was constructed by transferring the W-peptide (blue) from Evc2 to the C terminus of YFP-Smo. Yellow star: YFP tag.

(I) Localization of YFP-Smo and YFP-SmoW (green) at cilia (red) after transient expression in NIH/3T3 cells. Figure S4F shows that YFP-SmoW is localized to the EvC zone.

(J) Localization of YFP-Smo and YFP-SmoW at the EvC zone (orange) or the whole cilium (blue) in individual cells (circles) expressing varying amounts of the two proteins after transient transfection. Whole-cell YFP fluorescence is plotted for each cell; the black bar marks the mean fluorescence (or expression level) for each construct (n = 30).

(K) A mutant W-peptide carrying the A5 mutation that overlaps the FV motif (YFP-SmoW^A5) (E) and inhibits Hh signaling (F) cannot target Smo to the EvC zone. Scale bars: 2.5 μm and 0.5 μm in (B).

See also Figure S4.