The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors (original) (raw)
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Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes
The EMBO Journal, 2001
Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identi®cation of a novel 45 kDa protein that specifically binds Rab7GTP at its C-terminus. This protein contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. We named it RILP (Rab-interacting lysosomal protein) since it can be recruited ef®ciently on late endosomal and lysosomal membranes by Rab7GTP. RILP-C33 (a truncated form of the protein lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant-negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant-negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traf®c.
The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction
Biochemical and Biophysical Research Communications, 2005
Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.
Rab7: A Key to Lysosome Biogenesish V
2000
The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.
Rab7: A Key to Lysosome Biogenesis
Molecular Biology of the Cell, 2000
The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.
The rab7 GTPase resides on a vesicular compartment connected to lysosomes
Journal of Cell Science, 1995
Rab GTPases belong to the Ras GTPase superfamily and are key regulators of membrane traffic. Among them, rab7 has been localized on late endosomes of NRK cells but its function remains unknown. In order to investigate its role, we generated stable HeLa cell lines that express either wild type or a GTPase-defective mutant of rab7 in an inducible manner. A morphological analysis of the intracellular localization of these proteins was performed by confocal laser microscopy. Here we show that, in HeLa cells, rab7 is present on a vesicular compartment that extends from the perinuclear area to the cell periphery and shows only a partial colocalization with the cation-independent mannose 6-phosphate receptor, a marker for late endosomes. The topology of this compartment is dependent on the microtubule network since nocodazole treatment results in its scattering throughout the cytoplasm. In addition, we observed that, in contrast to the wild-type protein, a rab7 mutant with a reduced GTPase...
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.
Rabs grab motors: defining the connections between Rab GTPases and motor proteins
Current Opinion in Cell Biology, 2002
Rab GTPases and their effectors regulate membrane traffic by determining, along with cognate SNAREs, the specificity of transport vesicle docking and fusion steps. Recent studies have also implicated Rabs in the movement of these transport vesicles from their site of formation to their site of fusion, and several Rabs have been linked to specific microtubule- or actin-based motor proteins. Analyses
Journal of Biological Chemistry, 2012
Background: Lysosome-related organelles are a group of cell type-specific compartments with specialized functions, including melanosomes in melanocytes. Results: Cell type-specific Rab proteins, Rab32 and Rab38, colocalize and interact with the ubiquitous trafficking machinery in melanocytes. Conclusion: Rab32 and Rab38 cooperate with the ubiquitous trafficking machinery for melanosome biogenesis. Significance: Learning how lysosome-related organelles are built is key to understanding their biology. Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38-and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis. Lysosome-related organelles (LROs) 2 are a group of cell type-specific membrane-bound compartments with specialized functions (1-4). Examples of LROs include melanosomes, platelet-dense granules, lamellar bodies of lung type II epithelial cells, and lytic granules of cytotoxic T lymphocytes, and natural killer cells. The physiological functions of these organelles are diverse, from the production and storage of melanin pigments (melanosomes) and the regulation of platelet aggregation (dense granules) to killing virus-infected and tumor cells (lytic granules) (1-4). The LROs share common characteristics with lysosomes such as an acidic luminal pH, the presence of lysosome-associated membrane proteins in their limiting membrane, and a common biogenesis pathway (1-4). The close relationship between lysosomes and LROs is further demonstrated by certain human genetic disorders, including the Hermansky-Pudlak syndrome (HPS), that cause abnormalities in both organelle types (2, 4-6). HPS is a group of autosomal recessive diseases (OMIM 2033000) that are characterized by oculocutaneous albinism (hypopigmentation of eyes and skin) and prolonged bleeding that result from defects in the biogenesis of melanosomes and platelet-dense granules, respectively (2, 4-6). Some HPS patients present additional symptoms due to defects in other LROs, for instance defective lamellar bodies result in fatal pulmonary fibrosis and abnormal lytic granules cause immune deficiency (2, 4-6). In humans, different forms of the disease, named HPS-1 through HPS-9, have been associated with mutations in nine separate genes (2, 4-7). Orthologs of those genes and seven additional genes cause HPS-like disease in 16 mutant mouse strains (4-9). Several of those HPS genes encode proteins that assemble into stable complexes, named adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-1,-2, and-3 (4-10). The function of AP-3 in lysosome biogenesis has been well established; it characterizes a route for trafficking of integral membrane proteins, such as lysosome-associated membrane proteins, from early