Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling
Fig 4
N213 glycosylation partially compensates for N336 N-glycan loss.
A. The indicated mutants were expressed in Cl8 cells. Each of the mutant proteins migrated more quickly in SDS-PAGE than wild type Smo, but not as quickly as SmoNQ4 or NQ5. B. Glycosylation at N213 partially compensates for N336 glycan loss. The rescue reporter assay was performed as described in Fig 2A. Each of the indicated double mutants, with the exception of N213Q,N336Q, was able to rescue reporter gene expression in the smo knockdown background to a level similar to that of N336Q. dSmoN213Q,N336Q demonstrated a level of activity similar to dSmoNQ5. Significance was determined using Student’s t-test. C. N336Q-containing double mutants are retained in the ER. Cl8 cells expressing the indicated Smo proteins (anti-Myc, magenta), the Cal-EGFP-KDEL marker (green) and Hh were examined by immunofluorescence microscopy. Whereas wild type Smo reached the plasma membrane, the double mutants overlapped with the ER marker. ActinRed (red) marks F-actin. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). D. Treatment of lysates from WT or N213Q,N336Q expressing cells with deglycosylating enzymes reveals that the N213,336Q mutant is present in the EndoH sensitive ER fraction, arrowhead. E-E’. N336Q and N213Q,N336Q mutants have disulfide bond defects. Biotin-maleimide was used to tag free thiol groups in cellular lysates prepared from Cl8 cells expressing WT, N336Q, N213Q,N336Q and C320A dSmo proteins. WT dSmo is not well captured on NeutrAvadin beads (lane 2, bound). N-glycan mutants are captured similarly to the disulfide bond mutant C320A (lanes 3–5, bound), indicating that at least one disulfide bridge is disrupted by N-glycan loss. E’ shows the ratio of bound to unbound dSmo proteins normalized to kinesin. Relative binding was determined by densitometry analysis of two independent binding assays. C320A, which has an established disulfide bond defect served as positive control. It’s binding ratio was arbitrarily set to 1.0 and other values are shown relative to it. Error bars are provided to show the standard deviation between the two experiments. F. dSmoN213Q,N336Q fails to rescue smo knockdown in vivo. UAS-dsmoN213Q, N336Q was co-expressed with UAS-dicer and UAS-smo3’UTR using the nubbin-Gal4 driver. Its expression did not modify the smo knockdown phenotype (compare to Fig 2F). Multiple progeny were analyzed over two crosses and a representative wing is shown.