Abstract
Primary cilia are hairlike extensions of the plasma membrane of most mammalian cells that serve specialized signaling functions. To traffic properly to cilia, multiple cilia proteins rely on palmitoylation, the post-translational attachment of a saturated 16-carbon lipid. However, details regarding the mechanism of how palmitoylation affects cilia protein localization and function are unknown. Herein, we investigated the protein ADP-ribosylation factor-like GTPase 13b (ARL13b) as a model palmitoylated ciliary protein. Using biochemical, cellular, and in vivo studies, we found that ARL13b palmitoylation occurs in vivo in mouse kidneys and that it is required for trafficking to and function within cilia. Myristoylation, a 14-carbon lipid, is shown to largely substitute for palmitoylation with regard to cilia localization of ARL13b, but not with regard to its function within cilia. The functional importance of palmitoylation results in part from a dramatic increase in ARL13b stability, which is not observed with myristoylation. Additional results show that blockade of depalmitoylation slows the degradation of ARL13b that occurs during cilia resorption, raising the possibility that the sensitivity of ARL13b stability to palmitoylation may be exploited by the cell to accelerate degradation of ARL13b by depalmitoylating it. Together, the results show that palmitoylation plays a unique and critical role in controlling the localization, stability, abundance, and thus function of ARL13b. Pharmacological manipulation of protein palmitoylation may be a strategy to alter cilia function.
Highlights
Primary cilia are hairlike extensions of the plasma membrane of most mammalian cells that serve specialized signaling functions
Additional results show that blockade of depalmitoylation slows the degradation of ADPribosylation factor-like GTPase 13b (ARL13b) that occurs during cilia resorption, raising the possibility that the sensitivity of ARL13b stability to palmitoylation may be exploited by the cell to accelerate degradation of ARL13b by depalmitoylating it
Of 209 Tier 2 cilia proteins, 67 (32%) appeared in SwissPalm. These results suggest that palmitoylation of cilia proteins is widespread
Summary
Several cilia proteins have been reported to be palmitoylated and to require the lipid for proper trafficking to cilia. ARL13b C8S/C9S could be robustly expressed in 2E9, but it localized to cytoplasmic puncta (as was observed in transiently transfected IMCD3 cells in Fig. 1C), and it did not rescue the cilia length defect (Fig. 2, D and E) This result suggests that the nonpalmitoylated ARL13b cannot serve its usual function in support of cilia structure, probably because of its. ARL13b C8S/C9S Myr does partially rescue formation of active ARL3, but quantitative analysis reveals ϳ40% less staining intensity (Fig. 5, A and B) These data again suggest that myristoylation may not functionally substitute fully for palmitoylation on ARL13b. In the fish injected with Sco-C8S/C9S Myr, which exhibited dorsal curvature, a partial rescue of the cilia morphology is observed (Fig. 5E) These data support the conclusion that Sco-C8S/C9S Myr protein is less functionally active than WT ARL13b, but it has at least partial functional activity as a result of attachment of the myristoyl group. The data suggest that proteasomes are the predominant mode of degradation of ARL13b
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