Podocytes serve as the final barrier to urinary protein loss through a highly specialized structure called a slit membrane and maintain foot process and glomerular basement membranes. Podocyte injury results in progressive glomerular damage and accelerates sclerotic changes, although the exact mechanism of podocyte injury is still obscure. We focus on the staining gap (podocin gap) defined as the staining difference between podocin and synaptopodin, which are normally located in the foot process. In puromycin aminonucleoside nephrosis rats, the podocin gap is significantly increased (p < 0.05) and podocin is translocated to the cytoplasm on days 7 and 14 but not on day 28. Surprisingly, the gap is also significantly increased (p < 0.05) in human kidney biopsy specimens of poor-prognosis IgA nephropathy patients. This suggests that the podocin gap could be a useful marker for classifying the prognosis of IgA nephropathy and indicating the translocation of podocin to the cytoplasm. Next, we find more evidence of podocin trafficking in podocytes where podocin merges with Rab5 in puromycin aminonucleoside nephrosis rats at day 14. In immunoelectron microscopy, the podocin positive area was significantly translocated from the foot process areas to the cytoplasm (p< 0.05) on days 7 and 14 in puromycin aminonucleoside nephrosis rats. Interestingly, podocin is also translocated to the cytoplasm in poor-prognosis human IgA nephropathy. In this paper, we demonstrate that the translocation of podocin by endocytosis could be a key traffic event of critical podocyte injury and that the podocin gap could indicate the prognosis of IgA nephropathy.

Cell motility requires actin assembly mediated by Rac, a Rho family GTPase. In this issue, Palamidessi et al. (2008) show that Rab5-directed trafficking of Rac to the cell membrane is required for Rac-mediated actin assembly. Trafficking of Rac may influence the mode of cell migration during morphogenesis and cancer metastasis.

The objective of this study is to better understand the molecular mechanism of tumor invasion and metastasis, and isolating tumor metastasis-related genes. Two human lung adenocarcinoma cell lines AGZY-83a and Anip973 were studied. Anip973 was derived from AGZY-83a, but it manifested a very much higher metastatic potential than the parent line. Differential cDNA fragments were isolated by using the techniques of mRNA differential display, and analyzed by means of molecular cloning and sequencing. The expression of RAB5A gene in clinical samples of non-small cell lung cancer was determined by RT-PCR. There were significant differences between AGZY-83a and Anip973 in gene expression. Part of the differential cDNA fragments were cloned and sequenced. We found that there was over-expression of RAB5A gene in the Anip973 cell line. And there was over-expression of RAB5A gene in those samples of clinical lung cancer showing metastasis. In conclusion, the expression or over-expression of RAB5A gene was associated with the metastatic phenotype of Anip973. Probably, over-expression of RAB5A gene in non-small lung cancer may serve as a diagnostic marker for metastasis.

Endothelial cells are susceptible to infection by mycobacteria, but the endocytic mechanisms that mycobacteria exploit to enter host cells and their mechanisms of intracellular transport are completely unknown. Using pharmacological inhibitors, we determined that the internalization of Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Mycobacterium abscessus (MAB) is dependent on the cytoskeleton and is differentially inhibited by cytochalasin D, nocodazole, cycloheximide, wortmannin, and amiloride. Using confocal microscopy, we investigated their endosomal trafficking by analyzing Rab5, Rab7, LAMP-1, and cathepsin D. Our results suggest that MSM exploits macropinocytosis to enter endothelial cells and that the vacuoles containing these bacteria fuse with lysosomes. Conversely, the entry of MTB seems to depend on more than one endocytic route, and the observation that only a subset of the intracellular bacilli was associated with phagolysosomes suggests that these bacteria are able to inhibit endosomal maturation to persist intracellularly. The route of entry for MAB depends mainly on microtubules, which suggests that MAB uses a different trafficking pathway. However, MAB is also able to inhibit endosomal maturation and can replicate intracellularly. Together, these findings provide the first evidence that mycobacteria modulate proteins of host endothelial cells to enter and persist within these cells.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

[6]-Gingerol has been used to control diabetes and dyslipidemia; however, its metabolic role is poorly understood. In this study, [6]-gingerol increased adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation in mouse skeletal muscle C2C12 cells. Stimulation of glucose uptake by [6]-gingerol was dependent on AMPKα2. Moreover, both Inhibition and knockdown of AMPKα2 blocked [6]-gingerol-induced glucose uptake. [6]-Gingerol significantly decreased the activity of protein phosphatase 2A (PP2A). Inhibition of PP2A activity with okadaic acid enhanced the phosphorylation of AMPKα2. Moreover, the interaction between AMPKα2 and PP2A was increased by [6]-gingerol, suggesting that PP2A mediates the effect of [6]-gingerol on AMPK phosphorylation. In addition, [6]-gingerol increased the phosphorylation of Akt-substrate 160 (AS160), which is a Rab GTPase-activating protein. Inhibition of AMPKα2 blocked [6]-gingerol-induced AS160 phosphorylation. [6]-gingerol increased the Rab5, and AMPKα2 knockdown blocked [6]-gingerol-induced expression of Rab5, indicating AMPK play as an upstream of Rab5. It also increased glucose transporter 4 (GLUT4) mRNA and protein expression and stimulated GLUT4 translocation. Furthermore, insulin-mediated glucose uptake and Akt phosphorylation were further potentiated by [6]-gingerol treatment. This potentiation was not observed in the presence of AMPK inhibitor compound C. In summary, our results suggest that [6]-gingerol plays an important role in glucose metabolism via the AMPKα2-mediated AS160-Rab5 pathway and through potentiation of insulin-mediated glucose regulation.

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigen-presenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.

BACKGROUND

Salmonella enterica Typhimurium (ST) is a phagosomal pathogen that can infect placental trophoblast cells leading to abortion and severe maternal illness. It is unclear how the trophoblast cells promote profound bacterial proliferation.

METHODS

The mechanism of internalization, intracellular growth and phagosomal biogenesis in ST-infected human epithelial (HeLa), macrophage (THP-1) and trophoblast-derived cell lines (JEG-3, BeWo and HTR-8) was studied. Specific inhibitors were used to block bacterial internalization. Phagosomal maturation was determined by confocal microscopy, Western-blotting and release of lysosomal β-galactosidase by infected cells. Bacterial colony forming units were determined by plating infected cell lysates on agar plates.

RESULTS

ST proliferated minimally in macrophages but replicated profoundly within trophoblast cells. The ST-ΔinvA (a mutant of Salmonella pathogenicity island-1 gene effector proteins) was unable to infect epithelial cells, but was internalized by scavenger receptors on trophoblasts and macrophages. However, ST was contrastingly localized in early (Rab5⁺) or late (LAMP1⁺) phagosomes within trophoblast cells and macrophages respectively. Furthermore trophoblast cells (unlike macrophages) did not exhibit phagoso-lysosomal fusion. ST-infected macrophages produced IL-6 whereas trophoblast cells produced IL-10. Neutralizing IL-10 in JEG-3 cells accelerated phagolysomal fusion and reduced proliferation of ST. Placental bacterial burden was curtailed in vivo in anti-IL-10 antibody treated and IL-10-deficient mice.

CONCLUSIONS

Macrophages phagocytose but curtail intracellular replication of ST in late phagosomes. In contrast, phagocytosis by trophoblast cells results in an inappropriate cytokine response and proliferation of ST in early phagosomes.

CONCLUSIONS

IL-10 production by trophoblast cells that delays phagosomal maturation may facilitate proliferation of pathogens in placental cells.

Considering the large number of Rab proteins, only a few Rab-specific exchange factors have been found and characterized. Rab8 is involved in mediating polarized membrane traffic through reorganization of actin and microtubules. It is possible to use the yeast two-hybrid technique to find potential Rab activators. A human protein (Rabin8) and its rat equivalent (Rabin3) were found to bind Rab8 and function as nucleotide exchange factors for Rab8 but not for Rab3A and Rab5. Endogenous and ectopically expressed Rabin8 frequently colocalize with cortical actin. This association is increased by cytochalasin D and phorbol esters that also induced the translocation of both Rabin8 and Rab8 to lamellipodia-like structures. We also show that a GFP-fused Rabin8 behaves identically in this respect. Furthermore, coexpression of Rabin8 with the dominant negative mutant of Rab8 leads to translocation of Rabin8 onto vesicular structures enriched in cell protrusions, indicating that both Rab8 and Rabin8 are involved in mediating polarized membrane transport. This chapter presents a detailed description of the methods and protocols developed to find and characterize a Rab8-specific activator.

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

Endocytosis constitutes an essential process in the regulation of the expression of cell surface molecules and receptors and, therefore, could participate in the neural-glial interactions occurring during brain development. However, the relationship between endocytic pathways in astroglial cells under physiological and pathological conditions remains poorly understood. We analyzed the endocytosis and transcytosis processes in growing astrocytes and the possible effect of ethanol on these processes. Evidence demonstrates that ethanol affects endocytosis in the liver and we showed that ethanol exposure during brain development alters astroglial development changing plasma membrane receptors and surface glycoprotein composition. To study these processes we use several markers for receptor-mediated endocytosis, fluid phase endocytosis and non-specific endocytosis. These markers were labeled for fluorescence microscopy and electron microscopy. 125I-BSA was used to study the effect of ethanol on the internalization and recycling of this macromolecule. The distribution of several proteins involved in endocytosis (caveolin, clathrin, rab5 and beta-COP) was analyzed using immunofluorescence, immunoelectron microscopy and immunoblotting. Our results indicate that growing astrocytes have a developed endocytic system mainly composed of caveolae, clathrin coated pits and vesicles, tubulo-vesicular and spheric endosomes, multivesicular bodies and lysosomes. Ethanol exposure induces a fragmentation of tubular endosomes, decreases the internalization of 125I-BSA, alters the processing of internalized BSA, and decreases the levels of caveolin, clathrin, rab5 and beta-COP. These results indicate that ethanol alters the endocytosis and transcytosis processes and impairs protein trafficking in astrocytes, which could perturb astrocyte surface expression of molecules involved in neuronal migration and maturation during brain development.

Rab20 is a member of the Rab GTPase family, but its implication in macropinocytosis is unclear. We examined the spatiotemporal localization of Rab20 in RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab20. It was shown that Rab20 was transiently associated with macropinosomal membranes. During the early stage of macropinosome formation, Rab20 was slightly localized on the circular ruffles (macropinocytic cups), the precursor forms of macropinosomes, and was increasingly recruited to the newly formed macropinosomes. Although Rab20 was colocalized with Rab5 and Rab21 on macropinosomal membranes, the association of Rab20 with macropinosomes persisted even after the dissociations of Rab5 and Rab21 from macropinosomal membranes. Rab20 was then colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on macropinosomes for a while. Our data indicate that Rab20 is a novel component of macropinocytic pathway and functions at long-standing stages from early to late macropinosome maturation.

ALS2, the causative gene product for a number of recessive motor neuron diseases, is a guanine-nucleotide exchange factor for Rab5, and acts as a modulator for endosome dynamics. Recently, we have identified a novel ALS2 homolog, ALS2CL, which is highly homologous to the C-terminal half of ALS2. In this study, we investigate the molecular features of ALS2CL and its functional relationship with ALS2. A majority of ALS2CL is present as a homo-dimeric form, which can interact with the ALS2-oligomer, resulting in the formation of the large ALS2/ALS2CL heteromeric complex. In cultured cells, overexpressed ALS2CL is colocalized with ALS2 onto membranous compartments. Further, ALS2CL dominantly suppresses the endosome enlargement induced by a constitutively active form of ALS2, and results in an extensive perinuclear tubulo-membranous phenotype, which are dependent upon the ALS2CL-ALS2 interaction. Collectively, ALS2CL is a novel ALS2-interacting protein and is implicated in ALS2-mediated endosome dynamics.

Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.

The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

Mutations in the ALS2 gene has recently been linked to cases of juvenile amyotrophic lateral sclerosis, juvenile primary lateral sclerosis and ascending hereditary spastic paralysis. All reported mutations predict the production of truncated forms of Alsin suggesting a loss of function mechanism for these motor neuron disorders. Here we used the tetracycline-regulated expression system to overexpress the full-length and truncated forms of Alsin in different cell lines. Alsin overexpression caused severe phenotypic changes in monkey COS-7 cells including the enlargement and accumulation of early endosomes, impairment of mitochondria trafficking and fragmentation of the Golgi apparatus. Our results further demonstrate the requirement of the Alsin VPS9 domain for occurrence of the vacuolation process and the role of Alsin as a guanine nucleotide exchange factor for Rab5. Transfected human SW13 cells exhibited an unexpected centrosomal localization for Alsin that was linked to the presence of the c-terminal part of the protein. Immunofluorescence staining revealed a colocalization of Alsin with the centrosomal markers gamma-tubulin and A kinase anchoring protein (AKAP-450). Similar results were obtained with human LA-N-2 and SK-N-SH neuronal cells. Moreover endogenous Alsin was detected in a centrosome preparation purified from human cortical brain. Considering the crucial role of centrosome in the production of microtubules required for intracellular transport, these findings are of potential relevance for unravelling the disease mechanisms linked to Alsin mutations.

Skp1/Cul1/F-box (SCF)-type F-box proteins are a component of the Cullin-RING SCF ubiquitin E3 ligase, which is involved in numerous cellular processes. However, the function of non-SCF-type F-box proteins remains largely unknown. The Rab5-like small guanosine 5'-triphosphatase Vps21/Ypt51 is a key regulator of intracellular transportation; however, deletion of its isoforms, Ypt52 and Ypt53, results in only a modest inhibition of intracellular trafficking. The function of these proteins therefore remains largely elusive. Here we analyze the role of a previously uncharacterized non-SCF-type F-box protein, Roy1/Ymr258c, in cell growth and intracellular transport in Saccharomyces cerevisiae. Roy1 binds to Ypt52 under physiological conditions, and Skp1 is indispensable for the association of Roy1 with Ypt52. The vps21Δ yeast cells exhibit severe deficiencies in cell growth and intracellular trafficking, whereas simultaneous deletion of roy1 alleviates the defects caused by deletion of vps21. However, additional disruption of ypt52 in roy1Δvps21Δ cells largely suppresses the cell growth and trafficking observed in roy1Δvps21Δ cells. We demonstrate that Roy1 interacts with guanosine 5'-diphosphate-bound and nucleotide-free Ypt52 and thereby inhibits the formation of guanosine 5'-triphosphate-bound, active Ypt52. These results thus indicate that Roy1 negatively modulates cell viability and intracellular transport by suppressing Ypt52.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.

BACKGROUND

The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development.

RESULTS

To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation.

CONCLUSIONS

In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC-CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri-nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.

The intracellular transport and degradation of endocytosed mannosylated albumin (Man-BSA) was studied in cell cultures of rat liver endothelial cells by subcellular fractionation, fluorescence microscopy, and electron microscopy. The ligand used for subcellular fractionation experiments was labeled with 125I-labeled tyramine cellobiose or 131I-labeled tyramine cellobiose. The labeled degradation products are trapped in the degradative compartments and may therefore serve as markers for these compartments. Cell fractionation was performed using Nycodenz gradients. The cell fractionation experiments demonstrated that the ligand sequentially occupied three compartments of increasing density. After 15 min it was mainly found in large cisternal organelles that banded in the gradient at about 1.09 g/ml. These organelles were rab5 positive and showed a peripher distribution in the fluorescence microscope. Degradation of ligand started after 30-60 min and this coincided with its transfer to a electron lucent vesicle with a density of 1.12 g/ml. After>> 1 h, degradation products started to accumulate in perinuclear, electron-dense lysosomes that banded in the gradient at 1.15 g/ml. The density distribution of lysosomal beta-acetylglucosaminidase coincided with the densest organelle. The results obtained show that the degradation of ligand takes place sequentially in two types of lysosomes. The early lysosome is an electronlucent vesicle of low density, whereas the terminal lysosome is an electron-dense organelle with higher density and a more perinuclear distribution. The main degradation of the ligand takes place in the early lysosome. The transfer of ligand and degradation products from the early to the late lysosome is slow. Texas red-labeled ovalbumin (OVA) coincided with lysosomes labeled with OVA-Bodipy 24 h in advance only after 4-6 h.

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.