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Yu J
,
Chia J
,
Canning CA
,
Jones CM
,
Bard FA
,
Virshup DM
.
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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.
Figure 6.
ERGIC2 Interacts with WLS and Is Required for Wnt Secretion
(A) Knockdown efficiency of four independent ERGIC2 siRNAs in HeLa cells. HeLa cells were transiently transfected with 50 ng of pPGK-mWnt3A and 50 nM siRNA in 12-well plates. Total RNA was extracted 48 hr posttransfection. ERGIC2 mRNA abundance was determined by qPCR and normalized to GAPDH. Error bars represent SEM.
(B) Knockdown of ERGIC2 reduces expression of the Wnt target gene AXIN2. The same RNA samples as in (A) were examined by qPCR for AXIN2 abundance. Error bars represent SEM.
(C) Knockdown of ERGIC2 does not affect AXIN2 expression induced by WNT3A-conditioned medium. HeLa cells were treated with WNT3A-conditioned medium for 18 hr. β-catenin knockdown served as a positive control. Error bars represent SEM.
(D) Knockdown of ERGIC2 can be rescued by wild-type WLS but not mutant K537A overexpression. STF cells were transfected with 100 ng of pPGK-mWnt3A, the indicated amount of WLS plasmids (ng), and 50 nM siRNA in 12-well plates. Luciferase activity was measured 48 hr posttransfection as above. Error bars represent SD.
(E) Interaction of WLS with ERGIC2. STF cells were transfected with plasmids encoding GFP or GFP-ERGIC2, and wild-type WLS. Cells were lysed 50 hr posttransfection, and anti-GFP antibody was used for immunoprecipitation. The arrows indicate GFP-ERGIC2. Chicken polyclonal WLS antibody from GeneTex was used for the immunoblot.
(F) WLS rescues the effects of ERGIC2 morpholino in X. laevis embryos. Control embryos undergoing gastrulation at stage 10 display a dorsal blastopore lip. Embryos injected with ERGIC2 morpholino fail to initiate gastrulation at stage 10 and lack a dorsal blastopore lip (n = 70/80). WLS RNA overexpression results in premature blastopore closure by stage 10 (n = 72/80). The expression of WLS RNA is sufficient to rescue the ERGIC2 MO blastopore lip phenotype (n = 70/80) compared to control embryos.
See also Figure S6.
