Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway

Abstract

The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. Here we performed a genetic screen in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and identified the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, which are involved in the early secretory pathway of Wingless/Wnt. We provided genetic evidence demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. We found that Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, we showed that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, our data provide important insights into the early secretory pathway of Wg/Wnt.

Keywords: Baiser; Early secretory pathway; Sec22; Wingless/Wnt trafficking; p24.

Figure 1

RNAi screen identify Wingless secretion regulators. Schematic illustration of the Drosophila wing imaginal disc is shown in (A). ci (cubitusinterruptus, Green) and en (engrailed, Red) expression pattern in Drosophila wing disc are shown in (B). Wingless expression pattern is shown in (C) and a longitudinal section of Wg-producing cells from a Confocal Z-stack of Wing disc is shown in (D). Wild-type Wingless antibody staining is shown in (E). (F–I) Expression of UAS-Baiser^RNAi, UAS-CHOp24^RNAi, UAS-Eclair^RNAi and UAS-Sec22^RNAi were induced by enGal4, Wg accumulates in the posterior Wg-producing cells of wing disc. Wing discs are oriented anterior left, dorsal up.

Figure 2

Baiser is a direct cargo selector that escorts Wg. The Baiser insertion mutant detail and the real-time quantitative PCR results of Baiser homozygotes are shown in (A). Wing blade of wt (left) and Baiser (right) homozygous escaper (B). Wg accumulates (C′) in the Wg-producing cells bearing Baiser homozygous mutant clones marked by the absence of GFP (C). Wg (D″) co-localizes with Baiser (D′) and Eclair (D) in S2 cells. Wg can be immunoprecipitated with Baiser, CHOp24 but not Eclair (E). Over-expression of Baiser-V5 driven by enGal4 can rescue the Wg secretion defect caused by UAS-Baiser^RNAi (F), but not UAS-CHOp24^RNAi (G) or UAS-Eclair^RNAi (H). Wing discs are oriented anterior left, dorsal up.

Figure 3

Sec22 regulates Wg trafficking and is packaged together with Wg and p24. Two mutant alleles of Sec22 were generated through P-element-mediated imprecise excision (A). Drosophila S2 cell was fed with dsRNA of Sec22, Vps35, GFP and lacZ. Wg protein in the supernatant (S) and in the cell lysate (lysate) were detected (B). Wg accumulates (C′) in the Wg-producing cells bearing Sec22 homozygous mutant clones marked by the absence of GFP (C). Baiser-V5 (D′), Sec22-HA (D″) and Wg (D‴) co-localize precisely (D‴′) and DAPI is shown in (D).

Figure 4

p24 and Sec22 escort Wg for anterograde transportation. Wg, p24 and Sec22 are packaged together for Wg anterograde transportation from ER to Golgi. P24 proteins function as the cargo selector and Sec22 functions as the v-SNARE.