Figure 2.

UNC119 is a myristoyl-binding protein. (A) Stable LAP6-UNC119a and LAP6-UNC119b RPE cells were serum-starved, fixed, and immunostained for pericentrin (PCNT) and acetylated tubulin (AcTub). The arrow indicates the basal body and the arrowhead indicates the cilium. Bar, 3 μm. (B) LAP-tagged UNC119a and UNC119b were tandem affinity-purified from the stable cell lines used in A. The eluates were resolved by SDS-PAGE and visualized by silver staining. Labeled proteins were identified by mass spectrometry. (C) Table showing the top seven proteins predicted to be myristoylated that were identified by tandem affinity purification and mass spectrometry of the stable IMCD3 LAP-Unc119a line and their percent coverage. (D) LAP-tagged cystin amino acids 1–20 were tandem affinity-purified from RPE cells stably expressing LAP5-cystin 1–20. The eluate was resolved by SDS-PAGE and visualized by silver staining. UNC119a and UNC119b were identified by mass spectrometry. (E) Myc-tagged in vitro translated BBS1 or UNC119a were incubated with a C-terminally biotinylated peptide corresponding to cystin residues 2–20, either unmyristoylated (cystin) or myristoylated on its N terminus (Myr-cystin). Complexes were precipitated with streptavidin beads, resolved by SDS-PAGE, and analyzed by immunoblotting with a myc antibody. BBS1 was included as a negative control. (F) GST-UNC119a-binding reactions were performed with C-terminally Myc-tagged full-length NPHP3 as in Figure 1C except that either cystin or Myr-cystin peptide was included at 0.1, 1, 5, or 10 μM. DMSO was added as a control. (G, top panel) GST-UNC119a-binding reactions were performed as in Figure 1C with N-terminally Myc-tagged in vitro translated C5orf30. (Bottom panel) Peptide competition assay with GST-UNC119a and in vitro translated C5orf30 was performed as in the right panel of Figure 1F.