ARL-13/Arl13b associates with ciliary membranes via palmitoyl anchors. (A) N-terminal Pal motif ARL-13 variants (delPal and rPal) fail to localize to amphid (head) and phasmid (tail) cilia (c) and diffusely mislocalize in cell bodies (cb) and dendrites (d). C-terminal deletion ARL-13(del203–370) is also mislocalized, but membrane associations are maintained (arrows). Insets show high magnification images of PHA/B cilia. (B) HsArl13b ciliary localization requires an N-terminal Pal motif. Ciliated RPE1 cells transfected with GFP-tagged Arl13b(WT) or a C8S/C9S variant and costained for ciliary axonemes using acetylated α-tubulin antibody are shown. Merged images show that Arl13b(C8S/C9S) is highly diffuse and nonmembrane associated, with weak signals in cilia. The boxed regions are shown at high magnification in the insets. Dashed lines outline the cell. (C) Loss of Pal motif shifts HsArl13b to cytosolic fractions. A subcellular fractionation scheme for Flag-tagged Arl13b (WT and variants) transiently expressed in 293T cells is shown. Supernatants (Sup.) and pellets (Pell) were probed for Arl13b::Flag using Western blotting and an anti-Flag antibody. Calpain and Na⁺/K⁺ ATPase proteins mark cytosol and membrane fractions, respectively. Arl13b(WT) and Arl13b(1–357) pellet with Na⁺/K⁺ ATPase membrane proteins after 20,000 g spins, whereas Arl13b(C8S/C9S) remains in calpain-enriched cytosolic supernatants even after 200,000 g spins. (D) Arl13b is palmitoylated in 293T cells. 293T cells transiently transfected with GFP-tagged Arl13b(WT) or Arl13b(C8S/C9S) were metabolically labeled with [³H]palmitate, and proteins were separated by SDS-PAGE. The top panel shows a fluorograph; the bottom panel shows a Western blot with anti-GFP antibody. The arrow indicates palmitoylated Arl13b-GFP, and the asterisk indicates an unknown endogenous palmitoylated protein. Bars: (A) 5 µm; (B) 10 µm.