A juxtamembrane basolateral targeting motif regulates signaling through a TGF-β pathway receptor in Drosophila
Fig 4
Punt BLT activity is conserved in insects, is robust to mutations, and is a dominant basolateral determinant in the wing disc.
(A) Amino acid sequences of the juxtamembrane substitution constructs. The backbone protein is indicated with the incorporated BLT region indicated in brackets. Punt[Apis] and Wit[Apis] harbor juxtamembrane sequences from the respective honeybee Type II receptor ortholog. (B–D) BLT function is conserved in insect Punt proteins. A9-Gal4 driving the control UAS-Punt[Punt] protein revealed the absence of staining at or apical to the junctions (B). The corresponding sequence from honeybee also supported basolateral restriction of the Punt[Apis] protein (C). The corresponding Wit sequence substituted for Punt’s failed to provide BLT activity, with the Punt[Wit] protein detected in a pattern resembling Wit-GFP (D). See S5 Fig for 3-color staining profiles corresponding to B–D. (E–G) Mutation of the BLT leads to varied apicalization depending on the severity of the mutation. Representative staining and signal profiles for mutant versions of Punt with increasing number of changed amino acids expressed with A9-Gal4 are shown. The Punt-PTE variant generally showed a lack of staining in the apical domain (E and E′), similar to WT Punt. Mutation of the insect-conserved residues to Alanines (Punt-Conserved-Ala) led to moderate and spatially varied overlap with the aPKC apical marker (F and F′). Mutation of the entire 19 amino acids of the BLT region (Punt-19-Ala) abolished basolateral enrichment as shown by the overlap of Punt staining with Dlg and aPKC (G and G′). (H–J) Moving the Punt BLT to the juxtamembrane position of Wit caused basolateral restriction. Flag IF gives a nontrivial background signal enriched at the apical membrane (H). The control Wit[Wit] protein driven by A9-Gal4 showed enrichment at the apical membrane (I). Wit[Punt] displayed primarily basolateral distribution with many apical regions showing staining at background levels (J). A Wit-Δ19 construct had similar distribution to Wit, indicating that the juxtamembrane region of Wit does not control the apicobasal distribution (K). Double prime (′′) images are Fire false-color displays of the anti-Flag channel. See S6 Fig for profiles for images corresponding to H-K. Scale bars: 100 μm for primary images; 15 μm for prime and double prime microscope images. BLT, basolateral targeting; WT, wild-type.