Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling
Fig 2
N-linked glycans are required for dSmo trafficking and activity.
A. dSmoNQ5 does not signal in vitro. Cl8 cells were transfected with control or smo 5’UTR dsRNA, the ptcΔ136-luciferase reporter, pAc-renilla control, pAc-myc-smoWT or NQ5, and pAc-hh or empty vector control. Hh-induced reporter activity (gray bars) was ablated by knockdown of endogenous smo and rescued by dSmo cDNA lacking UTR sequence for wild type, but not for dSmoNQ5. B-B’. dSmoNQ5 demonstrates altered sub-cellular localization. Cl8 cells expressing Calreticulin-EGFP-KDEL ER marker (GFP-ER, green) and Myc-SmoWT or NQ5 in the presence or absence of Hh were imaged by immunofluorescence microscopy. Wild type dSmo (anti-Myc, magenta) localized to puncta that did not overlap with the ER marker in the absence of Hh, and translocated to the plasma membrane in response to Hh. The NQ5 mutant overlapped with the ER marker under both conditions. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right box). B’. GFP-ER colocalizes with V5 tagged BiP, Calnexin (Cnx) and Calreticulin (Crc). DAPI marks the nucleus. C-H. dSmoNQ5 does not signal in vivo. Transgenes encoding wild type (G) or NQ5 (H) dSmo proteins were expressed in the nubbin>dicer;smo3’UTR background (E). Whereas wild type Smo could rescue the loss of function phenotype induced by smo3’UTR, dSmoNQ5 could not (G-H compared to F and C-D, control). UAS-EGFP was expressed in the nubbin>dicer;smo3’UTR background and serves as a control for normalized transgene dosage (F). I. Wild type and NQ5 dSmo proteins are present at similar protein levels in wing imaginal disc tissue lysate. The dSmoN213Q,N336Q protein level is higher.