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Drug-resistant pathogenic and opportunistic bacteria pose a global health threat. Plants, particularly those employed in traditional (folk) medicine, may provide a source of antimicrobials compounds possessing novel mechanisms of action with which to combat current and emerging infectious diseases. However, experimental designs in ethnobotanical investigations of this type are complicated by several factors. Among these are chemotype variations among plants and plant parts, and potential antagonistic, additive or synergistic effects between plant compounds. These efforts are further hindered by a lack of standardized applicable chemical extraction methods in the field, which makes assessment and comparison of experimental results difficult. This overview thus seeks to provide insight into these complicating factors, give a brief historical survey of ancient investigations into antibiotics, and identify some of the strengths and weaknesses of the various modern methods and techniques routinely employed in ethnobotanical antibiotic research.

The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293 cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [18F]4-N-alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale and conventional silane-labeling radiochemical protocols. Preliminary positron-emission tomography (PET) imaging in mice showed the biodistribution of [18F]4-N-alkyl to have initial concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.

The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293 cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [(18)F]4-N-alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale and conventional silane-labeling radiochemical protocols. Preliminary positron-emission tomography (PET) imaging in mice showed the biodistribution of [(18)F]4-N-alkyl to have initial concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.

The fruits of saw palmetto have been used for the treatment of a variety of urinary and reproductive system problems. In this study we investigated whether the fruit extracts affect in vitro adipogenesis. Saw palmetto ethanol extract inhibited the lipid droplet accumulation by induction media in a dose-dependent manner, and it also attenuated the protein expressions of C-EBPα and PPARγ. Phosphorylation of Erk1/2 and Akt1 were also decreased by saw palmetto ethanol extract. This report suggests that saw palmetto extracts selectively affect the adipocyte differentiation through the modulation of several key factors that play a critical role during adipogenesis.

Dehydroleucodine, a known sesquiterpene lactone, and dehydroparishin-B, a new guaiane type sesquiterpene acid, were isolated from aerial parts of Artemisia douglasiana by chloroform extraction. We identified dehydroparishin-B as (7R)-2-oxo-guaia-1(10),3(4),5(6),11(13)-tetraen-12-oic acid by MS and NMR methods. We demonstrated that both dehydroparishin-B and dehydroleucodine blocked cell proliferation of B16 melanoma cells, but not normal murine Melan-A melanocytes, in a dose-dependent manner without affecting cell viability. We also found that both dehydroparishin-B and dehydroleucodine inhibited migration of B16 melanoma cells. These results suggest that dehydroleucodine and dehydroparishin-B could represent potential candidates for the treatment of metastatic melanomas.

Many species of the Papilionidae are unpalatable and protected from natural enemies by aristolochic acids (AAs) sequestered from their Aristolochiaceae food plants. Battus polydamas (Papilionidae, Troidinii) is an Aristolochia -feeding papiloinoid butterfly. The uptake of AAs and other secondary metabolites by B. polydamas larvae from Aristolochia c was examined. The AA composition of larva, pupa and imago of B. polydamas was determined by HPLC-PDA, HPLC-MS-MS and comparison with authentic samples. The plant is rich in AA-II which is accompanied by minor amounts of AAs I, III, IV, Ia, IIIa, IVa and IVb. All eight AA analogs plus four O-glucosides from the phenolic AAs Ia, IIIa, IVa and IVb were present in the insect extracts. The AA-analogs IVb, Ia O-glucoside, IVb O-glucoside are described for the first time. The larval haemolymph qualitatively reflects the AA-pattern of the plant and is also rich in AA-II. Analysis of the larval tissues and the osmeterial secretion shows that the phenolic AAs are significantly O-glucosylated. Except for AA-I, all the AAs are gradually catabolized into unknown compounds throughout the life stages of B. polydamas , being therefore much less abundant or absent in the imago. In the imago AA-II becomes undetectable whereas AA-I remains as the major AA. Results suggest that predominance of AA-I in the adult may be due to repressed degradation of this acid rather than selective sequestration from the plant or de novo synthesis by the insect. Some aspects related to aristolochic acid uptake and metabolism are discussed.

In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay. In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88,731 -73616863688). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (−/−) mast cells and examined FcϵRI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in −/− mast cells, including decreased basal FcϵRI expression, slowed FcϵRI internalization, elevated IgE + Ag–induced degranulation and IL-6 production, and the decreased ability of −/− cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface FcϵRI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen–activated −/− mast cells. By contrast, correction of these −/− phenotypes required a functional Vps9 domain. Thus, FcϵRI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

Activated epidermal growth factor receptor (EGFR) recruits intracellular proteins that mediate receptor trafficking and signaling. Rab5 and Rin1, a multifunctional protein with a Rab5 guanine nucleotide exchange factor domain, have been shown to regulate EGFR endocytosis (Barbieri et al., 2000; Tall et al., 2001). In this study, we demonstrate that overexpression of both dominant negative Rab5 (Rab5: S34N) and full-length Rin1 selectively block EGF activation of the Raf-Erk1/2 kinase pathway and EGF-stimulated incorporation of [ 3 H]thymidine into DNA without affecting the activity of JN and p38 kinase pathways. Expression of Rab5: S34N and Rin1 also block EGF induction of cyclin D1 transcription. In contrast, expression of Rin1: Δ, a natural splice variant of Rin1 lacking 47 amino acids in the Vps9p domain or Rab5, increase both activation of Raf-Erk1/2- and cyclin D1 transcription in response to EGF. These results indicate that Rab5 and the Raf/Erk signal transduction pathway play essential and selective roles in EGF-induced cell proliferation, and highlight a new function for Rab5 in EGF signaling.

Activation of receptor tyrosine kinases is a key feature in receptor signaling and membrane trafficking processes. In this study, we found that the insulin receptor tyrosine kinase activity is required for fusion between early endosomes. AG1024, a receptor tyrosine kinase inhibitor, blocked the in vitro endosome fusion in a concentration-dependent manner. We observed that Rab5: wild type partially rescued the fusion reaction, whereas Rab5: Q79L mutant fully rescued it. We also observed that treatment of cells with insulin receptor kinase inhibitor HNMPA-(AM)3 blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of insulin in intact cells. HNMPA-(AM)3 inhibitor also affected the endosomal co-localization of Rab5 and insulin receptor. However, the formation of Rab5: Q79L mutant-positive endosomes were not affected by the HNMPA-(AM)3 inhibitor. In addition, HNMPA-(AM)3 inhibitor affected the association of Rin1 to membrane upon insulin stimulation. Furthermore, Rin1 did not fully support endosome fusion in the presence of the AG1024 inhibitor. These results constitute the first evidence that, at least in part, the enzymatic activity of insulin receptor is required for the fusion events via the activation of Rab5.

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A “kinase-dead” EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

Pseudomonas aeruginosa , a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa . These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

Binding of growth factors and other cell-activating agents to cell surface receptors is known to trigger a complex series of events that initiate signal transduction. Ligand activation of many signal-transducing receptors accelerates receptor endocytosis. The classical view is that receptor internalization is primarily a mechanism of signal attenuation and receptor degradation, but more recent evidence suggests that internalization may mediate the formation of specialized signaling complexes on intracellular vesicles. The small Rab GTPases, master regulators of vesicle transport, can influence both receptor trafficking and receptor signaling pathways. They are localized to specific organelles and domains where they not only mediate vesicle docking and fusion but also influence the recruitment of effector proteins that mediate signal transduction and vesicle motility. It is interesting to speculate that extracellular stimuli contribute to the endocytosis of cell surface compo- nents for survival, defense, repair, storage and degradation. In addition, traffic regulation by external stimuli emphasizes the possible role in infection, aging, cancer and several degenerative diseases. Thus, receptor-mediated endocytosis regulation by small Rab GTPases not only provides a mechanism for attenuation of signaling but may also determine the quality of signal output by providing different combinations of downstream effectors at various endocytic compartments.

Rin1 has been shown to play an important role in endocytosis. In this study we demonstrated that depletion of Rin1 from the cytosol blocked the fusion reaction. More importantly, endosome fusion was rescued by the addition of Rin1 proteins depending on the presence of Rab5, and its effector EEA1. Furthermore, we found that Syntaxin 13, but not Syntaxin 7, was required by Rin1 to support endosome fusion. We also identified six mutations on the Vps9 domain of Rin1 that failed to rescue the fusion reaction. Two of them, Rin1: D537A and Rin1: Y561F mutants showed dramatic inhibitory effect on the fusion reaction, which correlate with their inability to properly activate Rab5 or to bind endosomal membranes. Taken together, our results suggest that specific residues on the Vsp9 domain of Rin1 are required for its interaction with Rab5, binding to the endosomal membranes and subsequent regulation of the fusion reaction.

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

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.

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E2 (PGE2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

1,25(OH)(2)D(3) antiproliferative properties are widely known. However, the molecular bases of these properties are only partially elucidated. Since 1,25(OH)(2)D(3) effectively arrests growth in many tumors and hyperplastic tissues whose growth is driven by co-expression of EGFR and its ligand TGF-alpha, it was hypothesized that 1,25(OH)(2)D(3) could affect the TGF-alpha/EGFR-autocrine growth loop. This study examined 1,25(OH)(2)D(3) regulation of EGFR-growth signals, using human epidermoid A431 cells, in which the overexpression of EGFR and TGF-alpha constitute the major autocrine mitogenic signal. 1,25(OH)(2)D(3) inhibited autocrine and EGF-induced A431 cell proliferation. Furthermore, 1,25(OH)(2)D(3) changed the cellular localization of both TGF-alpha and EGFR and inhibited ligand-dependent phosphorylation of EGFR and ERK1/2. In addition, 1,25(OH)(2)D(3) impaired autocrine and EGF-induced nuclear translocation of activated EGFR and, consequently, its binding to AT-rich DNA sequences and transcriptional activation of the cyclin D1 promoter. These results demonstrate that 1,25(OH)(2)D(3) alters EGFR membrane trafficking and down-regulates EGFR growth signaling.

Multivesicular endosomes (MVEs) are complex intracellular organelles that function in endocytosis. A major function of the endocytic pathway is to sort internalized macromolecules and membrane proteins. Appropriately sorted proteins, such as epidermal growth factor (EGF) receptor (EGFR), are incorporated into MVEs before transport to the lysosomal compartment, where degradation occurs. Thus, MVEs operate in the endosome-to-lysosome portion of the pathway. In yeast cells, where MVE formation has been extensively studied, the pathway terminates in the yeast vacuole, which is equivalent to the vertebrate lysosome. MVEs arise by invagination of the limiting membrane of an endosomal vesicle such that many small internal vesicles are formed, hence the term "multivesicular endosome." In part, the internalization and targeting of membrane proteins to the MVE involves ubiquitin, a small protein associated with protein degradation. In reticulocytes and certain antigen-presenting cells, MVEs are routed to the plasma membrane rather than the lysosome, releasing small vesicles called "exosomes" back into the extracellular space.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Summary: Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP-bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 A. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild-type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function. genesis 31:17–29, 2001. © 2001 Wiley-Liss, Inc.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is known to play an important role in signal transduction and membrane trafficking. We show that one enzyme responsible for PIP(2) production, phosphatidylinositol-4-phosphate 5-kinase type 1beta (PIPKbeta), is essential for epidermal growth factor receptor (EGFR)-mediated endocytosis. Expression of murine PIPKbeta in NR6 cells expressing EGFR strikingly increased receptor internalization. Moreover, the kinase was shown to form an immunoprecipitable complex with EGFR. Expression of either a truncated kinase or a kinase dead mutant inhibited EGFR endocytosis and also blocked the membrane recruitment of PIPKbeta and both clathrin light chain and dynamin. Our results delineate a novel mechanism by which PIPKbeta regulates receptor-mediated endocytosis and receptor tyrosine kinase membrane traffic.

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.

Transient expression of oncogenic Ha-Ras (Ras:V12) stimulates endocytosis. Using NIH3T3 cells expressing constitutively active protein kinase B/akt (PKB/akt) or kinase-dead PKB/akt, we show that PKB/akt mediates the stimulatory effect of Ras on endocytosis. Fluid phase endocytosis of horseradish peroxidase in cells expressing the constitutively active form of PKB/akt was elevated and insensitive to phosphatidylinositol 3-kinase inhibitors. However, expression of dominant negative Rab5:N34 blocked endocytosis in cells expressing the constitutively active form of PKB/akt. Transient expression of either Rab5:wt or Rab5:L79, a GTPase deficient mutant of Rab5, in cells expressing constitutively activated PKB/akt further increased endocytic rate. However, in cells expressing kinase-dead PKB/akt, endocytic rate was not affected by transient expression of Rab5:wt. Rab5:L79, on the other hand, increased endocytosis in cells expressing kinase-dead PKB/akt. Similar results were obtained using an in vitro endosome fusion reconstitution assay with cytosol prepared from cells expressing the activated PKB/akt or kinase-dead PKB/akt. Both Rab5:wt and Rab5:L79 stimulated endosome fusion when assayed in cytosol containing the activated PKB/akt, whereas only Rab5:L79 activated fusion when the assay utilized cytosol from kinase-dead expressing cells. We conclude that Ras activation of endocytosis requires both PKB/akt and Rab5 and that active kinase is required for activation Rab5.

GTP-binding proteins or GTPases are versatile cyclic molecular switches (Gilman, A.G. 1987 ; Bourne et al., 1990, 1992). In the past few years there has been an explosion of interest in unraveling the role of GTPases in membrane traffic (Balch et al., 1990; Bourne, H.R., 1988). GTP-binding proteins are classified into two broad families: ras-like monomeric GTP-binding proteins and the heterotrimeric G proteins. Evidences from genetics, immunolocalization, and functional assays has established that two subfamilies of monomeric GTP-binding proteins, the Rab and ARF subfamilies, are required for membrane traffic (Pfeffer, S.R., 1992; Rothman, J.E. and Orci, L., 1992). More recently, attention has turned to the role of heterotrimeric G proteins in membrane traffic (Balch et al., 1992; Barr et al., 1992). This review will focus solely on the recent data that suggest that heterotrimeric G proteins and monomeric GTP-binding proteins are involved in endosome-endosome fusion and in endocytic transport.