Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The IFT81-IFT74 complex acts as an unconventional RabL2 GTPase-activating protein during intraflagellar transport

ABSTRACT: Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70 amino acid long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2 suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

SUBMITTER: Dr. Esben Lorentzen

PROVIDER: S-SCDT-10_15252-EMBJ_2022111807 | biostudies-other |

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

The IFT81-IFT74 complex acts as an unconventional RabL2 GTPase-activating protein during intraflagellar transport.

Project description:Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70-amino-acid-long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

| S-EPMC10505919 | biostudies-literature

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

Project description: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 an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B 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.

| S-EPMC5556974 | biostudies-literature

The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3.

Project description:The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

| S-EPMC4255629 | biostudies-literature

The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport.

Project description:The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.DOI: http://dx.doi.org/10.7554/eLife.02419.001.

| S-EPMC4003483 | biostudies-literature

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46.

Project description:Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed ?-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal ?-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

| S-EPMC5418046 | biostudies-literature

Dimerisation-dependent GTPase reaction of MnmE: how potassium acts as GTPase-activating element.

Project description:MnmE, a Guanine nucleotide-binding protein conserved between bacteria and man, is involved in the modification of tRNAs. Here we provide biochemical and X-ray structural evidence for a new GTP-hydrolysis mechanism, where the G-domains of MnmE dimerise in a potassium-dependent manner and induce GTP hydrolysis. The structure in the presence of GDP-AlFx and potassium shows how juxtaposition of the subunits induces a conformational change around the nucleotide which reorients the catalytic machinery. A critical glutamate is positioned such as to stabilise or activate the attacking water. Potassium provides a positive charge into the catalytic site in a position analogous to the arginine finger in the Ras-RasGAP system. Mutational studies show that potassium-dependent dimerisation and GTP hydrolysis can be uncoupled and that interaction between the G-domains is a prerequisite for subsequent phosphoryl transfer. We propose a model for the juxtaposition of G-domains in the full-length protein and how it induces conformational changes in the putative tRNA-modification centre.

| S-EPMC1500855 | biostudies-literature

TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport.

Project description:Insulin stimulates glucose uptake in fat and muscle cells via the translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the cell surface. The signaling pathways linking the insulin receptor to GLUT4 translocation in adipocytes involve activation of the Rho family GTPases TC10alpha and beta. We report here the identification of TCGAP, a potential effector for Rho family GTPases. TCGAP consists of N-terminal PX and SH3 domains, a central Rho GAP domain and multiple proline-rich regions in the C-terminus. TCGAP specifically interacts with cdc42 and TC10beta through its GAP domain. Although it has GAP activity in vitro, TCGAP is not active as a GAP in intact cells. TCGAP translocates to the plasma membrane in response to insulin in adipocytes. The N-terminal PX domain interacts specifically with phos phatidylinositol-(4,5)-bisphosphate. Overexpression of the full-length and C-terminal fragments of TCGAP inhibits insulin-stimulated glucose uptake and GLUT4 translocation. Thus, TCGAP may act as a downstream effector of TC10 in the regulation of insulin-stimulated glucose transport.

| S-EPMC156759 | biostudies-literature

Crystal structure of the intraflagellar transport complex 25/27.

Project description:The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.

| S-EPMC3098482 | biostudies-literature

The IFT81-IFT74 complex enhances GTP hydrolysis to inactivate RabL2 during intraflagellar transport

Project description: Not available

| S-BSST1153 | biostudies-other

Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors.

Project description:Cilia serve as cellular antennae where proteins involved in sensory and developmental signaling, including G protein-coupled receptors (GPCRs), are specifically localized. Intraflagellar transport (IFT)-A and -B complexes mediate retrograde and anterograde ciliary protein trafficking, respectively. Using a visible immunoprecipitation assay to detect protein-protein interactions, we show that the IFT-A complex is divided into a core subcomplex, composed of IFT122/IFT140/IFT144, which is associated with TULP3, and a peripheral subcomplex, composed of IFT43/IFT121/IFT139, where IFT139 is most distally located. IFT139-knockout (KO) and IFT144-KO cells demonstrated distinct phenotypes: IFT139-KO cells showed the accumulation of IFT-A, IFT-B, and GPCRs, including Smoothened and GPR161, at the bulged ciliary tips; IFT144-KO cells showed failed ciliary entry of IFT-A and GPCRs and IFT-B accumulation at the bulged tips. These observations demonstrate the distinct roles of the core and peripheral IFT-A subunits: IFT139 is dispensable for IFT-A assembly but essential for retrograde trafficking of IFT-A, IFT-B, and GPCRs; in contrast, IFT144 is essential for functional IFT-A assembly and ciliary entry of GPCRs but dispensable for anterograde IFT-B trafficking. Thus the data presented here demonstrate that the IFT-A complex mediates not only retrograde trafficking but also entry into cilia of GPCRs.

| S-EPMC5341726 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data