Rab GTPases in organelle biogenesis and transport
The functional characterization of the small Rab5 GTPase in endocytosis has provided insights into the role of the conserved Rab family members as master regulators of organelle biogenesis and intracellular transport. Rab GTPases act as membrane organizers, specifying the structural and functional identity of organelles. In the endocytic pathway, Rab proteins occupy distinct membrane territories or "Rab-domains" linked by a binary switch system consisting of divalent effectors, GEFs and GAPs that sequentially activate and repress Rab GTPases along the pathway. The central role of Rab5 in endosome biogenesis has been revealed using a combination of an in vitro reconstitution system and in vivo studies that showed that Rab5 is the rate-limiting factor for the entire endocytic pathway.
Endocytosis is an essential process serving multiple key cellular functions, such as nutrient uptake, signal transduction, and defense against pathogens. The small GTPase Rab5 is an important regulator of endocytosis and early endosome function. A breakthrough into the functional mechanism of Rab5 came from the biochemical purification of a large set of interacting proteins by affinity chromatography (Christoforidis et al., 1999).
Complexity of the Rab5 interactome. Rab5 interactors purified from bovine brain cytosol by affinity chromatography on a GST-Rab5/GTPgS column were eluted (see total eluate), separated on a gel filtration column and the fractions were analyzed by SDS-PAGE followed by coommassie staining. The identity and function of several Rab5 effectors and regulators has been addressed in subsequent studies (Christoforidis et al., 1999).
We found that Rab5 regulates a large molecular network of different effectors and regulators (over 40 proteins), each contributing a specific function in vesicle formation, tethering, fusion and cytoskeleton-dependent motility of early endosomes. Subsequent work has shown that Rab5 effectors function cooperatively, i.e. the activity of one effector is essential for the activity of another. For example, the PI3-K Vps34 is required for the synthesis of PI(3)P and the recruitment of FYVE-proteins, such as EEA1, to the membrane of early endosomes through binding to PI(3)P. Divalent Rab effectors can also functionally connect different Rab proteins. Our group was the first to demonstrate that sequentially acting Rab GTPases are functionally coupled through a binary switch system employing divalent Rab effectors (Rabaptin-5, Rabenosyn-5) in the recycling pathway (Vitale et al. 1998; De Renzis et al. 2002). A Rab cascade regulates transport also in the degradative pathway (Rink et al., 2005).
Assembly of the Rab5-domain involves a complex cascade of molecular interactions including delivery of Rab5 to the membrane mediated by a GDI Displacement Factor (GDF, e.g. PRA-1; Sivar et al., Nature 2003), regulators of the nucleotide cycle (GEFs, e.g. Rabaptin-5/Rabex-5 complex, and GAPs, e.g. RN-Tre) and a series of Rab effectors regulating phosphoinositide metabolism (the hVps34 PI3-K that synthesizes PI(3)P), membrane tethering and fusion (EEA1, Rabenosyn-5/hVps45, Rabankyrin-5). The Rab5-domain assembly and function therefore depends on the cooperativity among Rab5 effectors and between Rab5 effectors and SNAREs. For example, the Rabaptin-5/Rabex-5 complex generates a positive feedback loop, enhancing the activity of Rab5 upon recruitment of the effector on the endosome membrane. The PI3-K activity of hVps34 expands the number of binding sites for Rab5 effectors which interact with PI(3)P, thus providing another positive feedback loop.
Compartamentalization of endosomes into Rab-domains
Rab5, Rab4 and Rab11, occupy distinct membrane domains or "Rab-domains" on early endosomes (Sönnichsen et al., 2000). Endosomes are thus organized in a modular system, each module or Rab-domain fulfilling a set of functions. A kind of “binary switch” system based on divalent Rab effectors, such as Rabaptin-5 (Vitale et al., 1998) and Rabenosyn-5 (De Renzis et al., 2002) connects one Rab GTPase to another, allowing the sequential transport of cargo between adjacent Rab-domains, e.g. from Rab5 to Rab4, from Rab5 to Rab7, etc. (Zerial and McBride, 2001).
Confocal images of Rab4-, Rab5- and Rab11-labeling on transferrin-filled endosomes. Rab5, Rab4 and Rab11 occupy distinct membrane domains called “Rab-domains” (see scheme on the right panel), each one fulfilling a different set of functions (adapted from Sönnichsen et al., 2000).
Are Rab machineries indefinitely maintained on membranes or can they disassemble in the course of cargo transport? Novel image analysis algorithms combined with fast live cell imaging revealed that the level of Rab5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Progression of cargo from early to late endosomes occurs via the replacement of Rab5 with Rab7, a process termed Rab conversion. These findings led us to propose a model whereby organelle transport entails the sequential flow of assembly and disassembly of Rab GTPases and their effectors on the membrane (Rink et al, 2005).
An endosome undergoing Rab5-to-Rab7 Conversion. This sequence of frames from a time-lapse movie (see Rab5/Rab7 video) shows an endosome in the perinuclear area containing fluorescently labelled LDL (DiD-LDL, red), where mRFP-Rab5 (green) is replaced with GFP-Rab7 (blue) (Rink et al., 2005).
Formation of macropinosomes in NIH3T3 cells transiently co-expressing YFP-Rabankyrin-5 and CFP-b-actin. Membrane ruffling driven by actin results in the formation of large, plasma membrane derived vesicles acquiring Rabankyrin-5. Some newly formed vesicles shed off Rabankyrin-5 over time and seem to regurgitate back to the plasma membrane via an actin comet-tail (Schantwinkel et al., 2004).
Reconstitution of Rab5- and SNARE-dependent membrane fusion
It has been proposed that SNAREs are sufficient to dock and fuse membrane in vitro. However, Rab GTPases and their effectors are recognized to be essential components acting at an earlier stage, i.e. tethering between membranes compatible for fusion and priming/pairing of SNAREs. We succeeded in the reconstitution of “synthetic” endosomes in vitro, made of a set of 17 recombinant proteins (in collaboration David Drechsel and the protein expression and purification facility) consisting of the Rab5 GTPase, its key regulators and effectors, early endosomal SNAREs. These vesicles could fuse with purified early endosomes or with each other in vitro, as measured by a bona fide content-mixing assay and morphological analysis by EM. Membrane fusion with SNAREs alone was almost undetectable and required cooperativity between Rab5 effectors and SNAREs, implying that the Rab5 machinery is a core component of membrane fusion and endosome biogenesis (Ohya et al., 2009).
Donor proteoliposomes containing syntaxin 13, VTI1A, syntaxin 6 and PRA1 and acceptor proteoliposomes with VAMP4 and PRA1 were incubated without cytosol (lane 1), with cytosol (lane 2) or with the indicated recombinant proteins (lanes 3–14). In lanes 13 and 14, '++' indicates the presence of 200 nM of proteins. Fusion efficiency was measured and the precise values are indicated above each column (Ohya et al., 2009).
We are currently establishing new assays to measure the forces and kinetics required to generate stable tethering between Rab5 effector proteins and membranes. We will investigate the transition from tethering to fusion using a combination of classical protein biochemistry, image-based assays and biophysical approaches.
Two Rab5 effector proteins, APPL1 and APPL2, localize with Rab5-GTP to what appears to be an endosomal compartment distinct from the canonical early endosome (Miaczynska et al., 2004). APPL proteins play a role in signal transduction: in HeLa cells, APPL proteins shuttle between endosomes and the nucleus, and are required for cell proliferation. Studies in zebrafish have shown that the regulation of Akt signalling depends on the endosomal localization of APPL1, which mediates the substrate specificity among Akt targets and selectively controls cell survival. Partitioning of Akt and selected effectors onto endosomal compartments represents a novel mechanism contributing to the specificity of signal transduction during vertebrate development.
We are currently characterizing the biogenesis and trafficking properties of APPL endosomes, as well as the coupling between endocytosis and signalling, in collaboration with Marta Miaczynska at the IIMCB in Warsaw .
HeLa cells were treated with Rh-EGF for 5 min, fixed, and stained with antibodies to APPL1 (green) or EEA1 (blue). We found that after 5 min of internalization, a fraction of Rh-EGF localized to fine punctate structures harboring APPL1 (adapted from Miaczynska et al., 2004).
Rab5 is necessary for the biogenesis of the endo-lysosomal system in vivo
Our in vitro studies demonstrated that Rab5 can assemble a multi-protein effector machinery on the endosome membrane that, together with SNARES, mediates efficient membrane tethering and fusion. In order to test directly the role of Rab5 in endosome biogenesis in vivo, we made use of state-of-the-art RNAi technology (in collaboration with Alnylam Pharmaceuticals) to knock down all three Rab5 isoforms in the adult mouse liver (Zeigerer et al., 2012). Loss of Rab5 below a certain threshold caused a dramatic reduction in the number of early endosomes, late endosomes and lysosomes. Additionally, animals depleted of Rab5 in the liver exhibited various metabolic phenotypes. We are currently using biochemistry, cell and systems biology methods to elucidate the interesting link between endocytosis and metabolism and characterize genes involved in the regulation of hepatocyte polarity.