A Novel Rab9 Effector Required for Endosome-to-TGN Transport (original) (raw)
Related papers
Rab9 functions in transport between late endosome to trans Golgi network
The EMBO Journal
Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In viro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelie may bear multiple rab proteins with different biological functions.
Characterization of Novel Rab6-Interacting Proteins Involved in Endosome-to-TGN Transport
Traffic, 2002
GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two-hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, Aƒ and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP-dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6-binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6Aƒ.
Regulation of Receptor Trafficking by Rab GTPases
2003
Human cells contain more than 60 Ras-like, Rab GTPases that are localized to the surfaces of distinct membrane-bound compartments. Rabs are master regulators of membrane traffic events: they can catalyze the formation of membrane microdomains, the collection of cargo into transport vesicles, the motility of vesicles along cytoskeletal tracks, the docking of vesicles with their targets, and the subsequent membrane fusion process. Rab protein function has broad implication for our understanding of human disease. For example, mutations in Rab27 lead to Griscelli syndrome due to defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in T cells. Other genetic diseases are caused by loss of function of multiple Rab proteins due to mutations in common Rab regulators. My laboratory studies the Rab9 GTPase that is essential for transport of mannose 6-phosphate receptors between late endosomes and the Golgi complex. Rab9 forms a distinct domain in late endosome ...
Journal of Biological Chemistry
Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.
The multiple roles of Rab9 in the endolysosomal system
Communicative & Integrative Biology, 2016
The small GTPase Rab9 has long been described as a protein that mediates endosome-to-trans-Golgi Network (TGN) transport, and specifically mannose-6-phospate receptor (MPR) recycling. However, studies have challenged this view by showing that Rab9 also is connected to sorting pathways toward the endolysosomal compartments. We recently characterized the spatio-temporal dynamics of Rab9 and, by using live cell imaging, we showed that it enters the endosomal pathway together with CI-MPR at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. More so, the Rab9 constitutively active mutant, Rab9Q66L, accumulates on late endosomes and promotes carrier formation at the TGN. Here, we discuss our findings in light of previous reports on Rab9 in the retrograde transport pathway.
The Journal of Cell Biology, 1994
Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carded to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ms-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged.
RhoBTB3: A Rho GTPase-Family ATPase Required for Endosome to Golgi Transport
Cell, 2009
Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3's N-terminal, Rho-related domain, and C-terminal sequences that are important for Rab9 interaction.9 Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the auto-inhibited RhoBTB3 protein to permit maximal ATP hydroysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.
Journal of Cell Biology, 2000
Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-defi-cient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures Ͻ 20 Њ C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.