ARL3 activation requires the co-GEF BART and effector-mediated turnover

ElMaghloob, Yasmin; Sot, Begoña; McIlwraith, Michael J.; Garcia, Esther Perez; Yelland, T.; Ismail, Shehab

doi:10.7554/elife.64624

Cited by 17 publications

(21 citation statements)

References 32 publications

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“…Likewise, we found no interaction between INPP5E and BART, a protein that cooperates with ARL13B as a co-GEF for ARL3 (data not shown) 53 .…”

Section: Inpp5e Binding To Arl13b Is Promoted By Cls2 Cls3 and Cls4mentioning

confidence: 63%

“…For this to happen, ARL3 must first be activated by a guanine nucleotide exchange factor (GEF) complex consisting of ARF-like 13B (ARL13B), an atypical small G-protein, and its cofactor Binder of ARL2 (BART) 17,18,48,[50][51][52][53] .…”

Section: Multiple Clss Target Inpp5e To Ciliamentioning

confidence: 99%

“…ARL13B regulates INPP5E in at least two different ways. First, ARL13B acts as a guanine nucleotide exchange factor (GEF) for ARL3, whose active GTP-bound form promotes dissociation of the PDE6D-INPP5E complex after it reaches the ciliary lumen [50][51][52][53] . Additionally, ARL13B directly interacts with INPP5E and is required for its ciliary retention 18,48,49 .…”

Section: Inpp5e Binding To Arl13b Is Promoted By Cls2 Cls3 and Cls4mentioning

confidence: 99%

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Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

Cilleros-Rodríguez

Martin-Morales

Barbeito

et al. 2022

Preprint

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Primary cilia are sensory membrane protrusions whose dysfunction causes diseases named ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, such as cilium stability, trafficking, signaling, or exovesicle release. Despite its key ciliary roles, how INPP5E accumulates in cilia remains poorly understood. Herein, we show that INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four ciliary localization signals (CLSs), the first two of which we newly discover: LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial redundancy between CLS1 and CLS4 explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 on the catalytic domain surface clarifies why CLS3 and CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations in INPP5E catalytic domain affect its ciliary targeting, and shed light on the mechanisms of action of each CLS. Thus, we find that CLS2 and CLS3 promote interaction with TULP3 and ARL13B, while downregulating CEP164 binding. On the other hand, CLS4 recruits PDE6D, RPGR and ARL13B, and cooperates with CLS1 in ATG16L1 binding. Lastly, we show INPP5E immune synapse targeting is CLS-independent. Altogether, we reveal unusual complexity in INPP5E ciliary targeting mechanisms, likely reflecting its multiple key roles in ciliary biology.

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“…Likewise, we found no interaction between INPP5E and BART, a protein that cooperates with ARL13B as a co-GEF for ARL3 (data not shown) 53 .…”

Section: Inpp5e Binding To Arl13b Is Promoted By Cls2 Cls3 and Cls4mentioning

confidence: 63%

Section: Multiple Clss Target Inpp5e To Ciliamentioning

confidence: 99%

Section: Inpp5e Binding To Arl13b Is Promoted By Cls2 Cls3 and Cls4mentioning

confidence: 99%

See 1 more Smart Citation

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

Cilleros-Rodríguez

Martin-Morales

Barbeito

et al. 2022

Preprint

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“…Furthermore, it was proposed that the ARL3 is kept in its GTP bound, active, form in complex with its co-GEF. 33 , 34 It is possible that the interplay between ARL13B, BART, and the ARL3 GTPase activating protein GAP (XRP2) generates a gradient or different local concentrations of ARL3-GTP within the primary cilium and immunological synapse. Both the primary cilium and the immunological synapse are known to be further compartmentalized 35 , 36 into specialized microdomains.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, it has been proposed that ARL3 is efficiently activated by its GEF ARL13B (guanine nucleotide exchange factora protein that facilitates activation of g-proteins by accelerating nucleotide exchange) only in the presence of another protein, binder of ARL2 (BART) protein which was termed a co-GEF. Furthermore, it was proposed that the ARL3 is kept in its GTP bound, active, form in complex with its co-GEF. , It is possible that the interplay between ARL13B, BART, and the ARL3 GTPase activating protein GAP (XRP2) generates a gradient or different local concentrations of ARL3-GTP within the primary cilium and immunological synapse. Both the primary cilium and the immunological synapse are known to be further compartmentalized , into specialized microdomains.…”

Section: Discussionmentioning

confidence: 99%

The Structural and Biochemical Characterization of UNC119B Cargo Binding and Release Mechanisms

et al. 2021

Self Cite

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Two paralogs of the guanine dissociation inhibitor-like solubilizing factors UNC119, UNC119A and UNC119B, are present in the human genome. UNC119 binds to N-myristoylated proteins and masks the hydrophobic lipid from the hydrophilic cytosol, facilitating trafficking between different membranes. Two classes of UNC119 cargo proteins have been classified: low affinity cargoes, released by the Arf-like proteins ARL2 and ARL3, and high affinity cargoes, which are specifically released by ARL3 and trafficked to either the primary cilium or the immunological synapse. The UNC119 homologues have reported differences in functionality, but the structural and biochemical bases for these differences are unknown. Using myristoylated peptide binding and release assays, we show that peptides sharing the previously identified UNC119A high affinity motif show significant variations of binding affinities to UNC119B of up to 427-fold. Furthermore, we solve the first two crystal structures of UNC119B, one in complex with the high affinity cargo peptide of LCK and a second one in complex with the release factor ARL3. Using these novel structures, we identify a stretch of negatively charged amino acids unique to UNC119B that may undergo a conformational change following binding of a release factor which we propose as an additional release mechanism specific to UNC119B.

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In vitro reconstitution of small GTPase regulation

et al. 2022

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Small GTPases play essential roles in the organization of eukaryotic cells. In recent years, it has become clear that their intracellular functions result from intricate biochemical networks of the GTPase and their regulators that dynamically bind to a membrane surface. Due to the inherent complexities of their interactions, however, revealing the underlying mechanisms of action is often difficult to achieve from in vivo studies. This review summarizes in vitro reconstitution approaches developed to obtain a better mechanistic understanding of how small GTPase activities are regulated in space and time.

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ARL3 activation requires the co-GEF BART and effector-mediated turnover

Cited by 17 publications

References 32 publications

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

The Structural and Biochemical Characterization of UNC119B Cargo Binding and Release Mechanisms

In vitro reconstitution of small GTPase regulation

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