SummaryWnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding -barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

Background: Neurogenic placodes are focal thickenings of the embryonic ectoderm that form in the vertebrate head. It is within these structures that the precursors of the majority of the sensory neurons of the cranial ganglia are specified. The trigeminal placodes, the ophthalmic and maxillomandibular, form close to the midbrain-hindbrain boundary and many lines of evidence have shown that signals emanating from this level of the neuraxis are important for the development of the ophthalmic placode.