Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling
Fig 3
N336Q impacts dSmo trafficking and activity.
A. N336 possesses an essential glycan modification. The rescue reporter assay was performed in smo knockdown Cl8 cells as in Fig 2. Each of the single N to Q mutants was able to rescue reporter gene activity in the smo knockdown background with the exception of N336Q. This mutant rescued to ~50% of the wild type activity. B. dSmoN336Q is mislocalized. Cl8 cells expressing Hh and the indicated dSmo proteins were imaged by immunofluorescence microscopy. Whereas each of the active single glycosylation site N to Q mutants (anti-Myc, magenta) reached the plasma membrane (indicated by F-actin stain, green) in Hh-expressing cells, dSmoN336Q failed to do so. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). C. SmoN336Q is retained in the ER. Myc-SmoN336Q expressed in Cl8 cells overlaps with the ER marker protein Calreticulin-KDEL-GFP. Scale bar is 5 μm (upper right). D. Treatment with deglycosylating enzymes demonstrates that the majority of dSmoN336Q is present in the EndoH sensitive, ER resident fraction (black arrowhead). WT and N213Q proteins are equally distributed between ER and post-ER fractions (white arrowhead).