Lipid-independent secretion of a Drosophila Wnt protein

Abstract

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.

FIGURE 1.

WntD secretion and signaling activity do not require Porcupine. A–D, third instar larval wing discs from heterozygous (A and C) or porcupine hemizygous zygotic mutant (B and D) animals in which UAS-wingless (A and B) or UAS-wntD (C and D) was expressed using dpp-GAL4. Wing discs were dissected and stained using antibodies against Wingless (Wg) or WntD protein and are displayed at 63× magnification with the dorsal-ventral axis oriented vertically and the anterior-posterior axis oriented horizontally. E–G, sagittal view of the posterior pole region of blastoderm stage embryos stained using antibodies against Dorsal to visualize the nuclear accumulation of Dorsal protein (ventral side, down; dorsal side, up). Arrowheads indicate the dorsal-most level of Dorsal nuclear accumulation in somatic cells, shown relative to the pole cells (PC) in wild type (E), wntD mutant (F), or porcupine maternal and zygotic mutant (G) embryos. H–J, cuticle preparations from wild-type embryos (H) or embryos in which UAS-wntD was overexpressed (OE) in the maternal germline using nanos-GAL4:VP16 in a wild type (I) or porcupine germline clone (por glc) mutant (J) background.

FIGURE 2.

WntD does not undergo lipid modification and is secreted at high levels. A, analysis of protein lipidation. S2 cells expressing either nothing (lane 1), FLAG-Wingless (lane 2), or FLAG-WntD (lane 3) were incubated with an azido-palmitic acid analog and secreted Wnt proteins were pulled down from the supernatant using anti-FLAG beads before reaction with phosphine-biotin via the Staudinger ligation. The incorporated azido-palmitic acid analog was detected with streptavidin-HRP (first panel). Wg (52 kDa) or WntD (34 kDa) protein levels were detected by stripping and reprobing the membrane with an anti-FLAG antibody conjugated to HRP (second panel). B, Coomassie Blue-stained protein gel showing purified FLAG-WntD protein in detergent (lane 1) and non-detergent (lane 2) conditions. C, deconvoluted mass spectrum of intact FLAG-WntD protein. D, amino acid sequence alignment between several Wnt family members, including Drosophila WntD, of residue serine 209 from mouse Wnt3a (boxed) and the surrounding sequence, which contains conserved cysteine residues (shaded). E, supernatant from S2 cells stably expressing FLAG-Wg (S2 cells, lane 1) or FLAG-WntD (S2 cells, lane 2) or L cells stably expressing FLAG-Wnt3a (L cells, lane 1) or FLAG-WntD (L cells, lane 2) was collected, centrifuged, and detected by Western blot with an anti-FLAG antibody. Total protein levels in each lane were confirmed to be similar by reversible Ponceau S staining of the membrane before anti-FLAG detection (data not shown).

FIGURE 3.

WntD secretion and signaling activity do not require Wntless. A–D, third instar larval wing discs from heterozygous (A and C) or wntless homozygous zygotic mutant (B and D) animals in which UAS-wingless (A and B) or UAS-wntD (C and D) was expressed using wingless-GAL4. Wing discs were dissected and stained using antibodies against Wingless (Wg) or WntD protein and are displayed at 63× magnification with the dorsal-ventral axis oriented vertically and the anterior-posterior axis oriented horizontally. The intense staining pattern of Wingless protein in wntless homozygous mutants is shown imaged under conditions identical to those used for heterozygous wing discs (left half of panel B) and at a lower exposure (right half of panel B)). E–G, sagittal view of the posterior pole region of blastoderm stage embryos stained using antibodies against Dorsal to visualize the nuclear accumulation of Dorsal protein (ventral side, down; dorsal side, up). Arrowheads indicate the dorsal-most level of Dorsal nuclear accumulation in somatic cells, shown relative to the pole cells (PC) in wild type (E), wntD mutant (F), or wntless maternal and zygotic mutant (G) embryos.

FIGURE 4.

WntD secretion requires the early secretory pathway component Rab1. Third instar larval wing discs in which UAS-wntD or UAS-wingless alone (A and C) or UAS-wntD or UAS-wingless together with dominant negative UAS-rab1^S25N (B and D) were expressed using dpp-GAL4. Wing discs were dissected and stained using antibodies against WntD or Wingless (Wg) protein and are displayed at 63× magnification with the dorsal-ventral axis oriented vertically and the anterior-posterior axis oriented horizontally. WntD staining in panel B is shown imaged at a 10-fold lower exposure time than panel A to show non-saturating conditions. Wingless staining in panels C and D are shown imaged under identical conditions.