Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. Here we show that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5(-) oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. We propose that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex.

Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotide exchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, a multivalent adaptor protein that also binds to Rab4, Rab5, and to domains present in gamma-adaptins and the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchange activity, thus generating GTP-bound, membrane-associated Rab5 that, in turn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex on endosomes. Although the Rabex-5.Rabaptin-5 complex is critical to the regulation of endosomal fusion, the structural determinants of this interaction are unknown. Likewise, the possible binding and covalent attachment of ubiquitin to Rabex-5, two modifications that are critical to the function of yeast Vps9p in endosomal transport, have not been studied. In this study, we identify the 401-462 and 551-661 coiled-coils as the regions in Rabex-5 and Rabaptin-5, respectively, that interact with one another. We also demonstrate that Rabex-5 undergoes ubiquitination and binds ubiquitin, though not via its proposed C-terminal CUE-like domain. Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising an A20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is important for ubiquitin binding and ubiquitination. Importantly, we demonstrate that the Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. These observations extend our understanding of the regulation of Rabex-5 by Rabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligase that binds ubiquitin and undergoes ubiquitination indicates that its role in endosome fusion may be subject to additional regulation by ubiquitin-dependent modifications.

Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.

After invasion of epithelial cells, Salmonella enterica Typhimurium resides within membrane-bound vacuoles where it survives and replicates. Like endocytic vesicles, the Salmonella-containing vacuoles (SCVs) undergo a maturation process that involves sequential acquisition of Rab5 and Rab7 and displacement toward the microtubule-organizing center. However, SCVs fail to merge with lysosomes and instead develop subsequently into a filamentous network that extends toward the cell periphery. We found that the initial centripetal displacement of the SCV is due to recruitment by Rab7 of Rab7-interacting lysosomal protein (RILP), an effector protein that can simultaneously associate with the dynein motor complex. Unlike the early SCVs, the Salmonella-induced filaments (Sifs) formed later are devoid of RILP and dynein, despite the presence of active Rab7 on their membranes. Kinesin seems to be involved in the elongation of Sifs. SifA, a secreted effector of Salmonella, was found to be at least partly responsible for uncoupling Rab7 from RILP in Sifs and in vitro experiments suggest that SifA may exert this effect by interacting with Rab7. We propose that, by disengaging RILP from Rab7, SifA enables the centrifugal extension of tubules from the Salmonella-containing vacuoles, thereby providing additional protected space for bacterial replication.

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits long-term Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.

Synthetic oligodeoxynucleotides (ODN) expressing non-methylated "CpG motifs" patterned after those present in bacterial DNA have characteristic immunomodulatory effects. CpG DNA is recognized as a pathogen-associated molecular pattern, and triggers a rapid innate immune response. CpG ODN are being harnessed for a variety of therapeutic uses, including as immune adjuvants, for cancer therapy, as anti-allergens, and as immunoprotective agents. The signal transduction pathway mediated by the engagement of CpG DNA with Toll-like receptor 9 (TLR9) is shared with other members of the TLR family. Recent studies demonstrate that formation and maturation of CpG DNA-containing endosomes are regulated by phosphatidylinositol 3 kinases and the Ras-associated GTP-binding protein, Rab5, which are essential for the initiation of TLR9-mediated signaling.

Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.

The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4. We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS-positive vesicles. However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.

Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport.

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.

Nerve growth factor (NGF) induces neurite outgrowth and differentiation in a process that involves NGF binding to its receptor TrkA and endocytosis of the NGF-TrkA complex into signaling endosomes. Here, we find that biogenesis of signaling endosomes requires inactivation of Rab5 to block early endosome fusion. Expression of dominant-negative Rab5 mutants enhanced NGF-mediated neurite outgrowth, whereas a constitutively active Rab5 mutant or Rabex-5 inhibited this process. Consistently, inactivation of Rab5 sustained TrkA activation on the endosomes. Furthermore, NGF treatment rapidly decreased cellular level of active Rab5-GTP, as shown by pull-down assays. This Rab5 down-regulation was mediated by RabGAP5, which was shown to associate with TrkA by coimmunoprecipitation assays. Importantly, RNA interference of RabGAP5 as well as a RabGAP5 truncation mutant containing the TrkA-binding domain blocked NGF-mediated neurite outgrowth, indicating a requirement for RabGAP5 in this process. Thus, NGF signaling down-regulates Rab5 activity via RabGAP5 to facilitate neurite outgrowth and differentiation.

Atg9 is a transmembrane protein essential for autophagy which cycles between the Golgi network, late endosomes and LC3-positive autophagosomes in mammalian cells during starvation through a mechanism that is dependent on ULK1 and requires the activity of the class III phosphatidylinositol-3-kinase (PI3KC3). In this study, we demonstrate that the N-BAR-containing protein, Bif-1, is required for Atg9 trafficking and the fission of Golgi membranes during the induction of autophagy. Upon starvation, Atg9-positive membranes undergo continuous tubulation and fragmentation to produce cytoplasmic punctate structures that are positive for Rab5, Atg16L and LC3. Loss of Bif-1 or inhibition of the PI3KC3 complex II suppresses starvation-induced fission of Golgi membranes and peripheral cytoplasmic redistribution of Atg9. Moreover, Bif-1 mutants, which lack the functional regions of the N-BAR domain that are responsible for membrane binding and/or bending activity, fail to restore the fission of Golgi membranes as well as the formation of Atg9 foci and autophagosomes in Bif-1-deficient cells starved of nutrients. Taken together, these findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.

BACKGROUND

The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.

RESULTS

Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.

CONCLUSIONS

Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.

Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5-to-Rab7 conversion is rather based on a newly characterized 'cut-out switch' analogous to an electrical safety-breaker. Both designs require cooperativity of auto-activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut-out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut-out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes.

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.

Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic.

The mammalian-infective bloodstream form of Trypanosoma brucei possesses a highly active endocytotic system. Evasion of the host immune response by T. brucei is dependent on antigenic variation of VSG (variant surface glycoprotein), but additional mechanisms for removal of surface-bound antibody also operate. Four Rab proteins, Tb (trypanosomal) RAB4, 5A, 5B and 11 are located to the endosomal system; TbRAB5A and TbRAB11 co-localize with internalized anti-VSG antibody and transferrin. A live cell assay was used to record a single cycle of endocytosis of anti-VSG IgG and transferrin, their subsequent degradation within the endosomal system and exocytosis of the products. TbRAB5A and TbRAB11 were involved in the overall process of endocytosis, degradation and exocytosis, whereas TbRAB5B and TbRAB4 were not implicated. The kinetics of anti-VSG IgG and transferrin recycling depend on the nucleotide state of TbRAB5A and TbRAB11. These data, together with previous work, suggest that IgG and transferrin initially enter a TbRAB5A sorting endosome and are most probably recycled subsequently via a TbRAB11-dependent step. Analysis of the recycled IgG and transferrin demonstrated extensive degradation of these recycled proteins. Degradation of transferrin was enhanced in cells expressing increased amounts of TbRAB5A or TbRAB11 with a Ser->>Asn mutation, but was decreased when active TbRAB11 was overexpressed. The extent of degradation of anti-VSG IgG was found to be unaffected by mutant Rab protein expression. The presence of an efficient mechanism for the removal of IgG bound to the external surface of T. brucei and its subsequent proteolysis within the recycling system suggests a role for this pathway in immune evasion.

Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.

It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were approximately 10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.