Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogene (original) (raw)
Related papers
Molecular biology of the cell, 2005
An integral part of cell division is the separation of daughter cells via cytokinesis. There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11- and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial...
The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking
Journal of cell science, 2002
Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells, overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of...
2005
There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11-and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission.
FERARI is required for Rab11-dependent endocytic recycling
Nature Cell Biology, 2020
Endosomal transport is essential for cellular organization and compartmentalization and cell-cell communication. Sorting endosomes provide a crossroad for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organisation of these processes requires membrane tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name FERARI (Factor for Endosome Recycling And Rab Interactions). The Rab binding module of FERARI consists of Rab11FIP5/RFIP-2 and rabenosyn-5/RABS-5, while the SNARE interacting module comprises VPS45 and VIPAS39/SPE-39. Unexpectedly, the membrane fission protein EHD1/RME-1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1/RME-1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways. 45 in E. coli. His-VPS-45 co-purified with SPE-39-GST (Figure 1a, Extended Data 3d). VPS-33.2, which together with SPE-39 is part of the CHEVI complex 27, 28 served as a positive control, while the HOPS specific VPS-33.1 did not bind SPE-39. These data indicate direct binding of SPE-39 to VPS-45. This interaction was confirmed by yeast-two-hybrid (Y2H) and pull-down experiments (Figure 1b, Extended Data 2d, d', Supplementary Table 3). Moreover, FERARI is conserved in mammalian cells (Figure 1c). VPS45 has been shown to directly interact with rabenosyn-5 29, 30. Similar to VPS45, rabenosyn-5 interacted with VIPAS39 (Figure 1b-e, Fig. Extended Data 1b, 1b', 2b, 2b', 2d, 2d'). Based on HOPS and CORVET, we speculated that FERARI might contain more subunits (Extended Data 1a). To identify additional components, we incubated the bacterially expressed SPE-39/VPS-45 complex with worm lysate and performed mass spectrometry (Supplementary Table 1), followed by Y2H assays and pulldowns from selected, streamlined candidates. We discovered two additional components of FERARI: the Epsin-homology domain (EHD) containing protein RME-1 and UNC-44 (Figure 1f, Supplementary Table 3). UNC-44 contains ankyrin motifs and a death domain. SPE-39 interacted well with RME-1 and the ankyrin motifs of UNC-44 (Figure 1f, Supplementary Table 3, Extended Data 1c, 1c',1d, 1d', 2a, 2a'). RME-1 is homologous to mammalian EHD1-4, and UNC-44 to ANK1-3. Since EHD1 has been shown to act at endosomes and EHD2 at the plasma membrane 31, 32 , we focused on EHD1. EHD1 co-precipitated and co-localized with rabenosyn-5 (Figure 1h, Extended Data 4a, b). Much less is known about ANK1-3. However, ANK3 appears to be the ANK protein most highly expressed in HeLa cells (Extended Data 4d). ANK3 co-precipitated with endogenous VIPAS39 (Figure 1g). Finally, endogenous VIPAS39 was pulled-down with all other Cell 3 Cell 2 Cell Spermatheca c b wild-type spe-39(RNAi) vps-45(RNAi) wild-type spe-39(RNAi) vps-45(RNAi) Cell 3 Cell 2 Cell 1 Cell Cell 2 Cell 1 30 40 50 20 % yolk-GFP wt spe-39(RNAi)
An ARF6/Rab35 GTPase Cascade for Endocytic Recycling and Successful Cytokinesis
Current Biology, 2012
Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo [1]. Membrane trafficking is crucial for animal cell cytokinesis [2-8], and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) [9-16] contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis [12]. Here, we report that the ARF6 GTPase [17, 18] negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.
Rab35 Regulates an Endocytic Recycling Pathway Essential for the Terminal Steps of Cytokinesis
Current Biology, 2006
Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes , and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid [2-4] and the septin SEPT2 . We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.
Rab11-FIP3 and FIP4 interact with Arf6 and the Exocyst to control membrane traffic in cytokinesis
The EMBO Journal, 2005
The dual Rab11/Arf binding proteins, family of Rab11interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.
Small GTPases, 2016
Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.