The CEP19-RABL2 GTPase Complex Binds IFT-B to Initiate Intraflagellar Transport at the Ciliary Base

Abstract

Summary Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium, and periodically enter the cilium suggesting a yet identified mechanism that triggers ciliary entry of IFT complexes. Using AP-MS purification of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP and the RABL2 GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex, and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes and thus initiates ciliary entry of IFT.

Homozygous mutation inCEP19,a gene mutated in morbid obesity, in Bardet-Biedl syndrome with predominant postaxial polydactyly

“…These new findings suggest that the loss of function by truncation at Tyr65* in our patients is detrimental for proper primary cilium function. Interestingly, Rabl2 knockout mouse displays features characteristic of ciliopathies that include infertility, obesity, polydactyly and retinal degeneration 39. As these phenotypes are reminiscent of BBS, we propose that loss of function of CEP19 can cause BBS due to failure to recruit RABL2B, mimicking a RABL2B knockout.…”

“…CEP19 was found in a stoichiometric complex with RAB2LB, a highly conserved guanosine triphosphatase (GTPase), which is recruited to the centriole by CEP19 at the base of cilia. RABL2B binds GTP, and then binds to intraflagellar transport B (IFT-B) complex, triggering its entry into the primary cilium 39. Thus, CEP19-RABL2B-IFT pathway is proposed as a new molecular mechanism for directed ciliary traffic, needed for primary cilium formation.…”

Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP

“…Examples of the loading efficiency can be found in the original paper. (Figure S4E in Kanie et al, 2017). …”

“…Typically, small GTPases have a very high affinity for guanine nucleotides, with dissociation constants in the nanomolar to picomolar range (Bos et al ., 2007), and therefore require guanine nucleotide exchange factors (GEFs) to lower their nucleotide affinity to allow for rapid activation. Notable exceptions include several highly conserved centrosomal/ciliary small GTPases, such as Rabl2 (Kanie et al ., 2017), ARL13B (Ivanova et al ., 2017), Ift27/Rabl4 (Bhogaraju et al ., 2011), and Arl6 (Price et al ., 2012), and many large GTPases, such as the dynamin family (Gasper et al ., 2009), which have lower affinities for GDP/GTP, with dissociation constants in the micromolar range. In this configuration, these GTPases can become activated without the use of GEFs via their intrinsic nucleotide exchange.…”

“…In this configuration, these GTPases can become activated without the use of GEFs via their intrinsic nucleotide exchange. Although small GTPases that show spontaneous exchange may have additional regulatory proteins, finding that a small GTPase has a micromolar affinity for GDP/GTP and is capable of spontaneous exchange may suggest the absence of a traditional GEF and suggest looking for other forms of GTPase regulatory factors (see Kanie et al [2017] for a notable example).…”