Palmitoylation of the ciliary GTPase ARL13b is necessary for its stability and its role in cilia formation

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.

Keywords: cilia; post-translational modification (PTM); protein palmitoylation; protein stability; protein targeting.

Figure 1.

Mouse ARL13b is palmitoylated. A, ARL13b is palmitoylated in mouse kidney and IMCD3 cells, as judged by the RAC assay. ARL13b is recovered in the presence, but not the absence, of hydroxylamine (HA). WB, Western blotting. B, mutant ARL13b (C8S/C9S), overexpressed in HEK cells, shows no palmitoylation. C, WT ARL13b, but not the C8S/C9S mutant, localizes to cilia (identified with acetylated tubulin) when transiently expressed in IMCD3 cells. The C8S/C9S mutant is found in a punctate pattern in the cytoplasm. Scale bars, 10 μm.

Figure 2.

Palmitoylation of ARL13b is required for formation of elongated cilia. A, schematic presentation of CRISPR-Cas9 strategy used to knock out Arl13b (KO). Two guide RNAs were used to remove exon 1 of mouse Arl13b. B, exon deletion was confirmed by PCR using primer pairs internal and external to the deletion segment. PCR product with internal primers are shown for control IMCD3 cells (lane 1) and for the knock-out cell line called 2E9 (lane 2). PCR products with external primers are shown for control IMCD3 cells (lane 3) and 2E9 (lane 4). C, Arl13b deletion was confirmed in 2E9 by Western blotting. D, ARL13b immunofluorescence in IMCD3, 2E9, and 2E9 cells reconstituted with ARL13b WT and ARL13b C8S/C9S. 2E9 cells show markedly shortened cilia, which is rescued by expression of WT but not C8S/C9S ARL13b. Scale bar, 5 μm. E, graph showing cilia length in IMCD3, 2E9, and reconstituted 2E9 cells. Lengths are significantly different when compared with 2E9+WT cells. **, p < 0.01; ****, p < 0.0001.

Figure 3.

Palmitoylation occurs in a variety of ARL13b mutants. A variety of mutants of ARL13b were prepared and stably expressed in 2E9 cells, including T35N and G28V, which are predicted to alter nucleotide binding; R79Q, which causes Joubert's syndrome; and a deletion of the carboxyl-terminal tail distal to amino acid 308 (see “Results” for details). A, palmitoylation of each mutant occurred normally, as demonstrated by uptake of the palmitate analog 17-ODYA. IP, immunoprecipitation. B, immunofluorescence of ARL13b mutant proteins stably expressed in 2E9 cells. All of the mutants form cilia, although shorter than those in 2E9+ARL13b WT. All of the mutants localized predominantly to cilia, except ARL13b 1–308, which localizes extensively to sites outside cilia. Scale bars, 5 μm. C, quantification of cilia length. Data for IMDC3, 2E9, 2E9+WT, and 2E9+C8S/C9S are replotted from Fig. 2 for comparison. Lengths are significantly shorter when compared with 2E9+WT. ***, p < 0.001; ****, p < 0.0001.

Figure 4.

Myristoylation, farnesylation, and monopalmitoylation confer cilia localization in ARL13b but do not fully restore cilia length and membrane binding. A, partial amino acid sequences of different acylation mutants, showing insertion-myristoylation sequence (ARL13b C8S/C9S Myr), farnesylation sequence (ARL13b C8S/C9S Far), and monopalmitoylation mutants C8S and C9S. B, immunofluorescence of 2E9 cells stably expressing the indicated mutants. All acylation mutants localized predominantly to cilia. Scale bars, 5 μm. C, all acylation mutants show shorter cilia than WT, but longer than the non-acylated C8S/C9S (data for 2E9 and 2E9+C8S/C9S are replotted from Fig. 2 for comparison). D, fractionation study shows that monopalmitoylation, myristoylation, and farnesylation all partially restore membrane association relative to WT. L, cell lysate in detergent-free lysis buffer after 900 × g spin; P, pellet; S, supernatant obtained after 20,000 × g spin of the cell lysate. E, cadherin is shown as a control for membrane-bound proteins, and GAPDH is shown as a control for soluble proteins. E, RAC assay confirms both the cysteines at positions 8 and 9 can be palmitoylated individually. F, chemical blockade of palmitoylation with 2BP treatment reduces cilia length (quantified in G) and ARL13b density in 2E9+WT (quantified in H) but not in 2E9+C8S/C9S Myr cells. *, p < 0.05; ns, not significant from t test when 2BP samples were compared with WT. Scale bar, 5 μm. I, lack of palmitoylation of ARL13b C8S/C9S Myr is confirmed by lack of 17-ODYA uptake. IP, immunoprecipitation. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Figure 5.

Myristoylation incompletely replaces functional role of dual palmitoylation despite restoring cilia localization. A, myristoylation only partially restores activation of ARL3. ARL13b is a guanine nucleotide exchange factor for ARL3. Active ARL3 is observed in the cilia of 2E9+WT cells, whereas it is absent in 2E9+ARL13b C8S/C9S, even in sites where the mutant ARL13b is expressed. This demonstrates a loss of functional activity in the palmitoylation-deficient ARL13b mutant. Active ARL3 is restored in 2E9+ARL13b C8S/C9S Myr, albeit at lower intensity. No active ARL3 is observed in 2E9+ARL13b 1–308, showing that the truncation mutant cannot activate ARL3. Scale bars, 5 μm. B, quantification of active ARL3 staining, normalized to level of ARL13b staining. C, zebrafish heterozygous for Sco^hi459, a null allele of the zebrafish ortholog of Ar13b, were mated. Homozygous null progeny displays ventral body curvature, whereas heterozygous and homozygous WT alleles display straight body. Injection of mRNA encoding Sco-WT rescues body curvature defect (Table 1), whereas Sco-C8S/C9S and Sco-C8S/C9S Myr does not. Injection of Sco-C8S/C9S Myr demonstrates some abnormal activity, as judged by the presence of occasional dorsal-curving fish. D, genotyping of Sco-C8S/C9S Myr injected dorsal curving fishes. Reference fishes were as follows: heterozygous fish (straight) (lane 1), wild-type fish (straight) (lane 2), homozygous mutant fishes (ventral body curvature) and test fishes (lanes 3 and 4), and homozygous mutant fishes (dorsal body curvature) (lanes 5–11). E, embryos 3 dpf were stained with antibody against acetylated tubulin to evaluate the pronephric cilia phenotype. In wild-type (uninjected control, straight) and rescued (Sco-WT–injected) embryos, bundles of cilia in the duct appear as a tight line (arrow). In the mutant embryos with ventral body curvature, only ragged discontinuous staining could be detected (arrow), suggestive of abnormal cilia morphology. In the Sco-C8S/C9S Myr–injected dorsal body curvature embryos, there is partial rescue of cilia formation morphology. *, p < 0.05.

Figure 6.

Palmitoylation of ARL13b increases stability and protects it from proteosomal degradation. A, steady-state levels of ARL13b C8S/C9S are reduced relative to WT, and levels are not increased by the addition of myristoylation. Expression levels of ARL13b were studied by Western blotting of lysates from 2E9 cell lines stably expressing the indicated protein. B, quantification of ARL13b expression levels. C, Sco-C8S/C9S expression in zebrafish is reduced relative to Sco-WT and is not rescued in Sco-C8S/C9S Myr. The blot shows levels of the GFP-tagged Sco-WT, Sco-C8S/C9S, and Sco-C8S/C9S Myr protein expression at 24 h postinjection. D, degradation rates of native ARL13b in IMCD3 cells are similar to rates of ARL13b-WT stably expressed in 2E9 cells. Rates were determined by cycloheximide (CYX) treatment, which blocked new protein synthesis for the indicated times, followed by Western blotting, and quantified in F. E, degradation rates of ARL13b C8S/C9S are much faster than ARL13b WT and are not rescued by myristoylation. Western blots are quantified in G. H, blockade of proteasome with MG-132 inhibited degradation of both ARL13b C8S/C9S and ARL13b C8S/C9S Myr, whereas blockade of lysosomal degradation with HCQ had only a minor effect. I, quantification of blots in H. CYX+HCQ and CYX+MG-132 were compared with CYX alone at 6 h. J, ARL13b expression level and cilia length are correlated. Data for expression level (derived from densitometry of Western blots from cells lysates and normalized to WT levels) are plotted with cilia lengths derived from analyses of fluorescent micrographs. When mutants that do not localize to cilia are excluded (i.e. ARL13b 1–308 and ARL13b C8S/C9S), a general correlation between expression level and cilia length is observed. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 7.

ARL13b palmitoylation is stable with little or no cycling. A, 2E9+WT cells were pulsed with 17-ODYA for 2 h and washed, and chased with palmitic acid-rich media containing cycloheximide (CYX) for 4 h. B, graph showing that the drop in normalized 17-ODYA intensity is comparable with ARL13b protein loss over 4 h (Fig. 6G). C, most palmitoylation of ARL13b occurs within 2 h of synthesis. 2E9+WT cells were treated with cycloheximide + MG132 for 2 h, followed by metabolic labeling with 17-ODYA, to assess palmitoylation of ARL13b that was >2 h postsynthesis. Minimal palmitoylation occurs under these conditions. D, graph shows normalized 17-ODYA intensity. IP, immunoprecipitation. **, p < 0.01; ***, p < 0.001.

Figure 8.

Depalmitoylation inhibitors slow degradation of ARL13b. A, 2E9+WT cells were heat-shocked at 42 ºC to induce cilia resorption, which is accompanied by reduced ARL13b at 30 min. The addition of blockers of deplamitoylation HDFP and palmostatin B (Palm B) mitigate the degradation of ARL13b. B, quantification of data. *, p < 0.05; ***, p < 0.001; ns, not significant.