一个新的人Rb26基因cDNA的克隆、表达及其增强细胞吞噬功能研究

Rab GTPase家族成员基因及其调节囊泡膜运输途径功能已有许多报道,但Rab26蛋白分子的结构和功能仍不清晰.通过生物信息学分析,并在人脑cDNA文库中克隆了一个新的Rab26基因全长cDNA序列,长1 656bp,与已发表的基因序列相比,在48～50位插入了GCC,在956位T被C取代,而在1 197位G被A取代.该序列包含一个771 bp完整的开放阅读框(ORE),编码256个氨基酸残基的Rab26蛋白,分子质量为27.9 ku(GenBank登录号No.AY646153),而非如以往报告的190个氨基酸残基.GFP荧光标记全长Rb26在哺乳动物细胞中表达显示,Rab26主要呈现在细胞膜状结构相联系的分布,发现该基因高表达能显著增强PE标记的红色异源蛋白质的吞噬.还应用逆转录.聚合酶链反应对多种人肿瘤细胞Rb26表达进行了研究,结果显示,Rab26在Acc2、SPC-A1,K562以及HeLa等肿瘤细胞株呈高表达,而在SMMOL/LC-7721、HepG2、Caco2等肝和肠上皮细胞株中则不表达,值得进一步深入研究.     

Mammalian suppressor of Sec4 modulates the inhibitory effect of Rab15 during early endocytosis

Rab15 is a novel endocytic Rab that counters the stimulatory effect of Rab5-GTP on early endocytic trafficking. Rab15 may interfere with Rab5 function directly by sequestering Rab5 effectors or indirectly through novel sets of effector interactions. To distinguish between these possibilities, we examined the effector binding properties of Rab15. Rab15 does not interact directly with the Rab5 effectors rabex-5 and rabaptin-5 in a yeast two-hybrid binding assay. Rather mammalian suppressor of Sec4 (Mss4) was identified as a binding partner for Rab15. Mss4 preferentially binds GDP-bound (T22N) and nucleotide-free (N121I) Rab15, consistent with the proposed role of Mss4 as a chaperone that stabilizes target Rabs in their nucleotide-free form. Mutational analysis of Rab15 indicates that lysine at position 48 (K48Q) is important for the binding of Rab15-GDP to Mss4. Moreover, the mutation K48Q counters the inhibitory phenotype of wild type Rab15 on receptor-mediated endocytosis in HeLa cells and homotypic endosome fusion in vitro without altering the relative amount of cell surface-associated transferrin receptor. Together, these data indicate a novel role for Mss4 as an effector for Rab15 in early endocytic trafficking.     

Identification and characterisation of novel Mss4-binding Rab GTPases

The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.     

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.     

The ubiquitous dermokine delta activates Rab5 function in the early endocytic pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking.     

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.     

Rab15 differentially regulates early endocytic trafficking

Rab GTPases play an important regulatory role in early endocytosis. We recently demonstrated that epitope-tagged Rab15 (HArab15) co-localizes with Rab4, -5, and -11 on early endosomal membranes in CHO cells (Zuk, P. A., and Elferink, L. A. (1999) J. Biol. Chem. 274, 22303-22312). To characterize the role of Rab15 in endocytosis, we prepared functional mutants of HArab15 and examined their effects on early endocytic trafficking. Wild-type HArab15 and its constitutively active, GTP-bound mutant (Q67L) reduce fluid phase and receptor-mediated endocytosis without affecting the rate of recycling from early endosomal compartments. Inhibition of early endocytosis appears to be due to a reduction in the rate of homotypic early endosome fusion. Conversely, mutations that constitutively inactivate HArab15 stimulate early endocytosis and the homotypic fusion of early endosomes in vitro. Unlike active forms of HArab15, constitutively inactive HArab15 mutants also affect recycling from early endosomal compartments. Moreover, the two constitutively inactive mutants, GDP-bound HArab15-T22N and the non-nucleotide binding mutant HArab15-N121I, differentially regulate the transit of fluid phase and receptor-mediated endocytic tracers through early/sorting endosomes. Together, these data suggest that HArab15 may counteract the reported stimulatory effect of Rab5 on early endocytosis. Consistent with this, overexpression of constitutively active HArab15-Q67L attenuates Rab5-stimulated endocytosis, whereas Rab5-stimulated endocytosis is augmented in cells overexpressing a constitutively inactive HArab15 mutant defective in guanine nucleotide binding (N121I). Our data indicate that HArab15 differentially regulates distinct steps in membrane trafficking through early/sorting and pericentriolar recycling endosomes.     

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.     

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Rab4 is an essential regulator of lysosomal trafficking in trypanosomes

Rapid endocytosis and recycling of surface proteins are important processes common to most nucleated eukaryotic cells. The best characterized membrane recycling routes are mediated by the small GTPases Rab4 and Rab11, but the precise roles that these pathways play have not been fully elucidated. The protozoan Trypanosoma brucei has a highly developed endocytic system that is similar to that found in metazoans, albeit with an accelerated rate of membrane turnover. We have used this organism to investigate the function of the trypanosome orthologue of Rab4 (TbRAB4) by a combination of RNA interference, microscopy, and quantitative trafficking assays. RNA interference-mediated suppression of TbRAB4 expression inhibited the growth of trypanosomes without affecting receptor-mediated endocytosis or ligand recycling. Ultrastructural analysis indicated a major defect in membrane transport events. The accumulation of fluorescent dextran, a fluid-phase marker, was blocked in cells lacking TbRAB4 protein. Since most fluid-phase markers are transported to the lysosome in T. brucei, the effects of TbRAB4 RNA interference on lysosomal function were investigated. By immunofluorescence, the major lysosomal protein p67 became progressively dispersed in cells lacking the TbRAB4 protein. Pulse-chase analysis demonstrated that initial proteolytic cleavage and glycan processing of p67 were unaffected but that cells failed to accumulate the later p67 proteolyzed products associated with the lysosome. To confirm the role of TbRAB4 in lysosomal trafficking, a constitutively active mutant, TbRAB4QL, was expressed. TbRAB4QL was closely associated with an enlarged multivesicular body that contained p67. In addition, cells expressing TbRAB4QL showed increased fluid-phase uptake when compared with the parental line. Taken together, these data suggest that TbRAB4 is involved in regulation of fluid-phase traffic to the lysosome in T. brucei but not in receptor-mediated endocytosis or recycling. These data have implications for the role of Rab4 in other cell systems.     

A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Rab5 Induces Rac-independent Lamellipodia Formation and Cell Migration

Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative Rab5 inhibits vesicle fusion. Here we show that introduction of active Rab5 in fibroblasts also induced reorganization of the actin cytoskeleton but not of microtubule filaments, resulting in prominent lamellipodia formation. The Rab5-induced lamellipodia formation did not require activation of PI3-K or the GTPases Ras, Rac, Cdc42, or Rho, which are all strongly implicated in cytoskeletal reorganization. Furthermore, lamellipodia formation by insulin, Ras, or Rac was not affected by expression of dominant negative Rab5. In addition, cells expressing active Rab5 displayed a dramatic stimulation of cell migration, with the lamellipodia serving as the leading edge. Both lamellipodia formation and cell migration were dependent on actin polymerization but not on microtubules. These results demonstrate that Rab5 induces lamellipodia formation and cell migration and that the Rab5-induced lamellipodia formation occurs by a novel mechanism independent of, and distinct from, PI3-K, Ras, or Rho-family GTPases. Thus, Rab5 can control not only endocytosis but also actin cytoskeleton reorganization and cell migration, which provides strong support for an intricate relationship between these processes.     

Rab6 interacts with the mint3 adaptor protein

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.     

Rab22a regulates the sorting of transferrin to recycling endosomes

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16 degrees C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.     

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.     

A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5.

The small GTPase Rab family, which cycles between GTP-bound active and GDP-bound inactive states, plays an important role in membrane trafficking. Among them, Rab5 is involved in early endocytic pathway, and several Rab5-binding proteins have been identified as regulators or effectors to coordinate the docking and fusion processes of endocytic vesicles. We describe a novel binding protein exhibiting unique biochemical properties for Rab5. The Rab5-binding protein enhances GDP-GTP exchange reaction on Rab5 but preferentially interacts with its GTP-bound form. Gel filtration and immunoprecipitation analyses indicate that the Rab5-binding protein functions as a tetramer composed of anti-parallel linkage of two parallel dimers. These results suggest that the newly identified protein may function as an upstream activator and/or downstream effector for Rab5 in endocytic pathway. Possible roles of the quaternary structure have been discussed in terms of the Rab5-mediated signaling.