Abstract
ABSTRACTINPP5E, a phosphoinositide 5-phosphatase, localizes on the ciliary membrane via its C-terminal prenyl moiety, and maintains the distinct ciliary phosphoinositide composition. The ARL3 GTPase contributes to the ciliary membrane localization of INPP5E by stimulating the release of PDE6D bound to prenylated INPP5E. Another GTPase, ARL13B, which is localized on the ciliary membrane, contributes to the ciliary membrane retention of INPP5E by directly binding to its ciliary targeting sequence. However, as ARL13B was shown to act as a guanine nucleotide exchange factor (GEF) for ARL3, it is also possible that ARL13B indirectly mediates the ciliary INPP5E localization via activating ARL3. We here show that INPP5E is delocalized from cilia in both ARL3-knockout (KO) and ARL13B-KO cells. However, some of the abnormal phenotypes were different between these KO cells, while others were found to be common, indicating the parallel roles of ARL3 and ARL13B, at least concerning some cellular functions. For several variants of ARL13B, their ability to interact with INPP5E, rather than their ability as an ARL3-GEF, was associated with whether they could rescue the ciliary localization of INPP5E in ARL13B-KO cells. These observations together indicate that ARL13B determines the ciliary localization of INPP5E, mainly by its direct binding to INPP5E.
Highlights
ARF/ARL and RAB families of small GTPases function as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state to regulate various cellular processes, such as membrane trafficking
The data presented in this study, together with those presented in our previous study, support the direct role of ARL13B at least within cilia, independently of its ARL3-guanine nucleotide exchange factor (GEF) activity, for the following reasons: (i) the interaction of INPP5E with ARL13B via its ciliary targeting sequence (CTS) is required for its retention on the ciliary membrane but not for its entry into cilia (Qiu et al, 2021); Fig. 6
(ii) the localization of ARL13B on the ciliary membrane is required for the ciliary localization of INPP5E (Fig. 6E, J, K); (iii) analyses using ARL13B mutants, which were demonstrated to lack ARL3GEF activity (Ivanova et al, 2017), indicate that the ciliary localization of INPP5E does not correlate with ARL3-GEF activity of the ARL13B mutants but roughly correlates with their direct binding to INPP5E (Fig. 6E–I, K); and (iv) the GTP-locked mutant, ARL3(Q71L), did not restore the ciliary localization of INPP5E when expressed in ARL13B-KO cells (Fig. 6N, Q), it did restore the localization of INPP5E in ARL3-KO cells (Fig. 2K, N)
Summary
ARF/ARL and RAB families of small GTPases function as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state to regulate various cellular processes, such as membrane trafficking. Primary cilia are microtubule-based protrusions on the surface of various eukaryotic cells, which act as cellular sensors of extracellular mechanical stimuli, such as fluid flow, and of signaling molecules, such as the Hedgehog (Hh) family of morphogens (Bangs and Anderson, 2017; Gigante and Caspary, 2020). To achieve these sensory functions, specific proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are located on the ciliary membrane and in the cilioplasm (Ishikawa et al, 2012; Mick et al, 2015). The importance of these structures has been indicated by the fact that mutations in the genes encoding TZ components cause a broad spectrum of congenital disorders, generally called the ciliopathies (Braun and Hildebrandt, 2017; Reiter and Leroux, 2017); these include Meckel syndrome, Joubert syndrome (JBTS), Bardet-Biedl syndrome (BBS), and nephronophthisis
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