Dimerization of small GTPase Rab5

Rab proteins are small GTPases, localized to distinct cellular compartments, regulating specific steps of intracellular membrane trafficking. One member of the Rab family, Rab5, consists of three isoforms, Rab5a, Rab5b, and Rab5c, which have been shown to play an important role in early events of endocytosis. Using the yeast two-hybrid system, we have identified several cytosolic proteins that interact with the Rab5b Q79L (decreasing intrinsic and GTPase-activating protein-stimulated GTPase activities). Surprisingly, most positive clones were Rab5b or Rab5c, indicating that Rab5 could dimerize among extra-isoforms in the yeast two-hybrid system. In vitro and in vivo chemical cross-linking assays demonstrated that lipid-unmodified wild-type Rab5b purified from Escherichia coli, wild-type Rab5b, or a dominant active form Rab5b Q79L expressed in human 293T cells dimerized. Furthermore, the same assays using a Rab5b R81A substitution mutant showed that the Arg81 in the Switch II region [the second GTP/GDP binding motif (residues 74-93)] was essential for Rab5b dimerization. These results suggest that Rab5 isoforms can be dimerized depending upon the GTP-bound conformation, but independent upon a lipid modification.     

Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport.

Presenilin 1 (PS1) mutations account for the majority of early-onset dominant cases of familial Alzheimer's disease. Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticulum, Golgi apparatus, nuclear region and vesicular structures. These proteins include from seven to nine putative transmembrane domains, with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm. We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11, a small GTPase belonging to the Ras-related superfamily. Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11, a region including the fourth GTP-binding domain. Considering the implication of Rab proteins in vesicular transport pathways, the PS-Rab11 inter-action suggests that PSs might be involved in amyloid precursor protein vesicular routing.     

Characterization of a Rab11-like GTPase,EhRab11, of Entamoeba histolytica

The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.     

Recycling endosomes and TLR signaling--the Rab11 GTPase leads the way

The ability of Toll-like receptors (TLRs) to activate innate immunity depends on their transport to pathogen-containing organelles. In this issue of Immunity, Husebye et?al. (2010) report that delivery of TLR4 to phagosomes occurs via a recycling endosome intermediate, which is controlled by the GTPase Rab11a.     

ALS2, a novel guanine nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics

ALS2 mutations account for a number of recessive motor neuron diseases including forms of amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. Although computational predictions suggest that ALS2 encodes a protein containing multiple guanine nucleotide exchange factor (GEF) domains [RCC1-like domain (RLD), the Dbl homology and pleckstrin homology (DH/PH), and the vacuolar protein sorting 9 (VPS9)], the functions of the ALS2 protein have not been revealed as yet. Here we show that the ALS2 protein specifically binds to small GTPase Rab5 and functions as a GEF for Rab5. Ectopically expressed ALS2 protein localizes with Rab5 and early endosome antigen-1 (EEA1) onto early endosomal compartments and stimulates the enlargement of endosomes in cultured cortical neurons. The carboxy-terminus of ALS2 protein carrying a VPS9 domain mediates not only the activation of Rab5 via a guanine-nucleotide exchanging reaction but also the endosomal localization of the ALS2 protein, while the amino-terminal half containing RLD acts suppressive in its membranous localization. Further, the DH/PH domain in the middle portion of ALS2 protein enhances the VPS9 domain-mediated endosome fusions. Taken together, the ALS2 protein as a novel Rab5-GEF, ALS2rab5GEF seems to be implicated in the endosomal dynamics in vivo. Notably, a feature common to eight reported ALS2 mutations among motor neuron diseases is the loss of VPS9 domain, resulting in the failure of Rab5 activation. Thus, a perturbation of endosomal dynamics caused by loss of ALS2 rab5GEF activity might underlie neuronal dysfunction and degeneration in a number of motor neuron diseases.     

A mutation in Rab38 small GTPase causes abnormal lung surfactant homeostasis and aberrant alveolar structure in mice

The chocolate mutation, which is associated with oculocutaneous albinism in mice, has been attributed to a G146T transversion in the conserved GTP/GDP-interacting domain of Rab38, a small GTPase that regulates intracellular vesicular trafficking. Rab38 displays a unique tissue-specific expression pattern with highest levels present in the lung. The purpose of this study was to characterize the effects of Rab38-G146T on lung phenotype and to investigate the molecular basis of the mutant gene product (Rab38^cht protein). Chocolate lungs exhibited a uniform enlargement of the distal airspaces with mild alveolar destruction as well as a slight increase in lung compliance. Alveolar type II cells were engorged with lamellar bodies of increased size and number. Hydrophobic surfactant constituents (ie, phosphatidylcholine and surfactant protein B) were increased in lung tissues but decreased in alveolar spaces, consistent with a malfunction in lamellar body secretion and the subsequent cellular accumulation of these organelles. In contrast to wild-type Rab38, native Rab38^cht proteins were found to be hydrophilic and not bound to intracellular membranes. Unexpectedly, recombinant Rab38^cht proteins retained GTP-binding activity but failed to undergo prenyl modification that is required for membrane-binding activity. These results suggest that the genetic abnormality of Rab38 affects multiple lysosome-related organelles, resulting in lung disease in addition to oculocutaneous albinism.     

Distinct roles of Rab11 and Arf6 in the regulation of Rab11-FIP3/arfophilin-1 localization in mitotic cells

Rab11 family interacting protein 3/arfophilin-1 is a dual effector of Rab11 and Arf6 and exhibits Rab11-dependent localization to recycling endosomes in interphase. Furthermore, FIP3 undergoes dynamic redistribution to the intercellular bridge during cytokinesis. However, regulation of FIP3 redistribution and its local function by Rab11 and Arf6 has remained controversial. In this study, we developed a procedure for detecting endogenous FIP3, Arf6, and Rab11 and determined that FIP3 is localized near the intercellular bridge during cytokinesis, and to the Flemming body (the midbody) immediately before abscission; Rab11 is localized near the intercellular bridge, but not to the Flemming body; and Arf6 is localized to the Flemming body. Time-lapse analyses showed that FIP3 is transported to the intercellular bridge during cytokinesis, together with Rab11; before abscission, FIP3 becomes localized to the Flemming body, where Arf6 is already present. After abscission, FIP3 and Arf6 are incorporated into one of the daughter cells as a Flemming body remnant. Based on these observations, we propose that FIP3 localization to recycling endosomes in interphase and their transport to the intercellular bridge during cytokinesis depend on Rab11, and targeting of FIP3-positive endosomal vesicles to the Flemming body in the abscission phase depends on Arf6.     

Rab11 is a useful tool for the diagnosis of microvillous inclusion disease

Microvillous inclusion disease (MVID) is a congenital condition presenting with intractable diarrhea. Biopsies demonstrate abnormal apical PAS and CD10 staining in surface enterocytes correlating with the presence of characteristic cytoplasmic inclusions. MVID has been linked to mutations in myosin Vb, important in apical membrane recycling. Rab11 associates with myosin Vb in vesicle membranes and is also integral in recycling plasma membrane components. The authors performed Rab11 immunostaining on biopsies from 7 MVID cases, 10 normal small intestines, and 10 with chronic enteritis. In MVID cases, Rab11 showed diffuse apical cytoplasmic staining of surface enterocytes in a pattern similar to PAS and CD10, which was absent in all the 20 control cases. Ultrastructural examination confirmed localization to the external surface of MVID cytoplasmic inclusions. Rab11 staining may be a useful adjunct in MVID diagnosis and the results support that myosin Vb dysfunction is important in the pathogenesis of MVID.     

Characterization of a Rab7-like GTPase,EhRab7: a marker for the early stages of endocytosis in Entamoeba histolytica

We have identified a 667 base pair Rab7-like cDNA (EhRab7) from Entamoeba histolytica. The EhRab7 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 24.5 kDa. Alignment of EhRab7 with other Rab proteins demonstrated that EhRab7 shared significant homology at the amino acid level with Rab7-like proteins from a number of other eukaryotes, suggesting that EhRab7 is a Rab7 homolog for E. histolytica. Using immunofluorescence microscopy, EhRab7 was demonstrated to be associated with early fluid-phase endosomes (<30 min) and secretory vesicles. The association of EhRab7 with early endosomes disappeared 1 h after their formation. Immunofluorescence microscopy also revealed that this GTPase did not colocalize significantly with phagosomes nor with markers for other organelles including the endoplasmic reticulum, Golgi and late endosomes. These results, together with the known function for Rab7 in other systems, suggest that EhRab7 is bound to vesicles, and that it may participate in vesicle docking and fusion in secretory events, and in the early stages of fluid-phase endocytosis in E. histolytica.     

Identification and characterization of a novel variant in the highly conserved catalytic center of Rab11a

Small GTPases of the Rab family regulate vesicular traffic and distribution of proteins in different cell types. Rab11a is a member of this GTP hydrolyzing protein class and acts as a mediator of insulin stimulated translocation of the glucose transporter GLUT4 in peripheral tissues including heart and skeletal muscle. Here we report on Rab11a Q70R, a mutation in the catalytic center of Rab11a, observed in the cardiomyoblast cell line H9c2. Analysis of GTPase activity showed that Rab11a Q70L acts as a classical constitutive active mutant. Interestingly, the GTPase activity of Rab11a Q70R was not significantly different from the enzymatic activity of the Rab11a Q70 wild type protein. We therefore conclude that the glutamine residue of Rab11a at position 70 is not strictly essential for GTPase activity of this protein in contrast to Ras and other Rab proteins.     

Roles for the recycling endosome, Rab8, and Rab11 in hantavirus release from epithelial cells

Hantavirus structural proteins are believed to localize to intracellular membranes often identified as Golgi membranes, in virus-infected cells. After virus budding into the Golgi luminal space, virus-containing vesicles are transported to the plasma membrane via trafficking pathways that are not well defined. Using the New World hantavirus, Andes virus, we have investigated the role of various Rab proteins in the release of hantavirus particles from infected cells. Rabs 8 and 11 were found to colocalize with Andes virus proteins in virus infected cells and when expressed from cDNA, implicating the recycling endosome as an organelle important for hantavirus infection. Small interfering RNA-mediated downregulation of Rab11a alone or Rab11a and Rab11b together resulted in a decrease in infectious virus particle secretion from infected cells. Downregulation of Rab8a did not alter infectious virus release but reduction of both isoforms did. These data implicate the recycling endosome and the Rab proteins associated with vesicular transport to or from this intracellular organelle as an important pathway for hantavirus trafficking to the plasma membrane.     

Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis

Rab GTPases and their effectors play important roles in membrane trafficking between cellular compartments in eukaryotic cells. In the present study, we examined the roles of Rab11B and its effectors in insulin secretion in pancreatic β-cells. In the mouse insulin-secreting cell line MIN6, Rab11 was co-localized with insulin-containing granules, and over-expression of the GTP- or the GDP-bound form of Rab11B significantly inhibited regulated secretion, indicating involvement of Rab11B in regulated insulin secretion. To determine the downstream signal of Rab11-mediated insulin secretion, we examined the effects of various Rab11-interacting proteins on insulin secretion, and found that Rip11 is involved in cAMP-potentiated insulin secretion but not in glucose-induced insulin secretion. Analyses by immunocytochemistry and subcellular fractionation revealed Rip11 to be co-localized with insulin granules. The inhibitory effect of the Rip11 mutant was not altered in MIN6 cells lacking Epac2, which mediates protein kinase A (PKA)-independent potentiation of insulin secretion, compared with wild-type MIN6 cells. In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. The present study shows that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic β-cells.     

Rab5 GTPase对膀胱癌细胞自噬及增殖的调控

目的探讨Rab5 GTPase对膀胱癌细胞自噬及增殖的影响。方法采用实时定量PCR检测60例膀胱癌组织以及相应正常膀胱组织中Rab5 mRNA的表达水平,并检测Rab5 mRNA在膀胱癌细胞系及正常膀胱细胞系中的表达情况。通过pcDNA3.1-Rab5转染膀胱癌细胞系BIU-87、RT4过表达Rab5;免疫荧光、Western blot检测自噬相关蛋白LC3Ⅱ/Ⅰ、LC3斑点数目变化情况以及自噬相关蛋白Vps34的表达变化;采用CCK-8检测膀胱癌细胞的增殖改变情况。结果Rab5 mRNA在膀胱癌组织中的表达高于正常膀胱组织,Rab5 mRNA在膀胱癌细胞中的表达高于正常膀胱细胞系,差异有统计学意义(P<0.05);免疫荧光显示过表达Rab5后LC3荧光斑点明显增多,且过表达Rab5促进膀胱癌细胞中Vps34的表达增加及LC3-Ⅰ向LC3-Ⅱ的转化;过表达Rab5能促进膀胱癌细胞增殖(P<0.05)。结论膀胱癌组织中Rab5 mRNA的表达高于正常膀胱组织,Rab5提高膀胱癌细胞自噬水平并促进膀胱癌细胞增殖,且与自噬相关蛋白Vps34相关。     

Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.     

Bcr/Abl P190 interaction with Spa-1, a GTPase activating protein for the small GTPase Rap1

The Bcr/Abl oncogene is responsible for the development of Ph-chromosome positive acute lymphoblastic leukemia and chronic myelogenous leukemia in humans. Previous studies demonstrated that Bcr/Abl expression is associated with elevated levels of activated Rap1, a small GTPase. Levels of activated Rap1 are determined by a balance between GTPase activating and G-nucleotide exchange factor activity. We show that Bcr/Abl forms a protein-protein complex with Spa-1, a GTPase activating protein for Rap1, both in COS-1 cells as well as in primary lymphoblastic leukemia cells from a transgenic P190 BCR/ABL mouse model. The interaction between Spa-1 and P190 did not affect the tyrosine kinase activity of P190, nor did Spa-1 become phosphorylated on tyrosine as a result of the interaction. P190 and Spa-1 co-localized to peripheral actin structures in primary lymphoblasts and expression of Spa-1 in the leukemic lymphoblasts decreased the migration of these cells. The binding of Bcr/Abl to Spa-1 may cause aberrant subcellular location of Spa-1 and affect migration of these cells.