The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition>> 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs.

We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabS(Rab7) mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41-dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabS(Rab7) are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabS(Rab7), early endosomal Rab5s-RabA and RabB-reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabS(Rab7) is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabS(Rab7) detectable in the same-frequently the less motile-structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.

The small GTPases Rab5 and Rab7 are important organisers of endosome formation and maturation. In addition, they orchestrate the trafficking of cargo through the endosomal pathway. A crucial event during maturation of endosomes is the replacement of the early organiser Rab5 with the late organiser Rab7 in a process called Rab conversion. Rab conversion is a prerequisite for late events, chief among them the fusion of matured endosomes with the lysosome. Recent work identifies members of the Sand1/Mon1 protein family as crucial factors during this process. Here, we present an analysis of the function of the Drosophila ortholog of mon1/sand1, Dmon1. We found that loss of function of Dmon1 results in an enlargement of maturing endosomes and loss of their association with Rab7. The enlarged endosomes contain Notch and other trans-membrane proteins as cargo. We report the first electron microscopy analysis of Dmon1 cells in a metazoan and extend the analysis of the endosomes in mutant cells. Our results suggest that the phenotype can be explained by the loss of function of Rab7. Moreover, the endosomes of Dmon1 cells mature normally in many aspects, despite the loss of association with Rab7. Surprisingly, we did not observe overactive or ectopic signalling through receptors such as Notch and RTKs in Dmon1 mutant cells, as would have been expected because of the accumulation of receptors in the maturing endosomes of these cells. This was the case even when receptor uptake into intraluminal vesicles was suppressed.

The multifunctional vacuole is the largest organelle in plant cells, and many proteins are transported to and stored in this organelle; thus, the vacuole has great physiological and agronomical importance. However, the molecular mechanism and regulation of plant vacuolar traffic remain largely unknown. In this study, we demonstrate that multiple vacuolar trafficking pathways operate in plants. RAB5 and RAB7 are evolutionarily conserved subfamilies of Rab GTPase, whose animal and yeast counterparts regulate vacuolar/endosomal trafficking in a sequential manner. Functional analyses of a putative activating complex for RAB7 indicated that this complex is responsible for maturation from RAB5- to RAB7-positive endosomes in plant cells. Moreover, these machinery components are recruited to a more complex trafficking network. Mutations in RAB5 and RAB7 conferred counteracting effects on the vti11 mutant. Furthermore, impairment of RAB5- and RAB7-dependent pathways differentially affected the transport of distinctive cargos. These results indicate that plants have developed a complex vacuolar transport system distinct from that of nonplant systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels.

The early events in Crimean-Congo hemorrhagic fever virus (CCHFV) have not been completely characterized. Earlier work indicated that CCHFV likely enters cells by clathrin-mediated endocytosis (CME). Here we provide confirmatory evidence for CME entry by showing that CCHFV infection is inhibited in cells treated with Pitstop 2, a drug that specifically and reversibly interferes with the dynamics of clathrin-coated pits. Additionally, we show that CCHFV infection is inhibited by siRNA depletion of the clathrin pit associated protein AP-2. Following CME entry, we show that CCHFV has a pH-dependent entry step, with virus inactivation occurring at pH 6.0 and below. To more precisely define the endosomal trafficking of CCHFV, we show for the first time that overexpression of the dominant negative forms of Rab5 protein but not Rab7 protein inhibits CCHFV infection. These results indicate that CCHFV likely enters cells through the early endosomal compartment.

The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6-7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of < or =0.5-microm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.

Mutations in the ALS2 gene cause a number of recessive motor neuron diseases, indicating that the ALS2 protein (ALS2/alsin) is vital for motor neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for Rab5 (Rab5GEF) and is involved in endosome dynamics. However, the spatiotemporal regulation of the ALS2-mediated Rab5 activation is unclear. Here we identified an upstream activator for ALS2 and showed a functional significance of the ALS2 activation in endosome dynamics. ALS2 preferentially interacts with activated Rac1. In the cells activated Rac1 recruits cytoplasmic ALS2 to membrane ruffles and subsequently to nascent macropinosomes via Rac1-activated macropinocytosis. At later endocytic stages macropinosomal ALS2 augments fusion of the ALS2-localized macropinosomes with the transferrin-positive endosomes, depending on the ALS2-associated Rab5GEF activity. These results indicate that Rac1 promotes the ALS2 membranous localization, thereby rendering ALS2 active via Rac1-activated endocytosis. Thus, ALS2 is a novel Rac1 effector and is involved in Rac1-activated macropinocytosis. All together, loss of ALS2 may perturb macropinocytosis and/or the following membrane trafficking, which gives rise to neuronal dysfunction in the ALS2-linked motor neuron diseases.

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells.

Exo-endocytotic cycling of synaptic vesicles (SVs) is one of the most intensely studied membrane trafficking pathways. It is governed by sets of conserved proteins including Rab GTPases. Long considered to define the identity and composition of a subcellular organelle, it has become increasingly evident that multiple Rabs co-exist on intracellular compartments, each contributing to its membrane organization and specialised function. Indeed, we have recently demonstrated that at least 11 distinct Rab proteins co-exist on highly purified SVs. These include Rabs involved in exocytosis (Rab3a/b/c and Rab27b) and intermediates of SV recycling such as early endosomes (Rab4, Rab5, Rab10, Rab11b and Rab14). Interestingly, we found that while two of these proteins, namely Rab3a and Rab27b, exhibited differential cycling dynamics on SV membranes; they played complementary roles during Ca(2+)-triggered neurotransmitter release. The implications of these findings in the SV trafficking cycle are discussed.

Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases. DOI: http://dx.doi.org/10.7554/eLife.01623.001.

Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.

Ras is a master GTPase switch controlling multiple signal transduction cascades in the regulation of cell proliferation and differentiation. Rab5 is a local GTPase switch that is localized on early endosomes and controls early endosome fusion. This study demonstrates that the catalytic domain of p120 GTPase-activating protein (GAP), a well known Ras GAP, is able to interact physically with Rab5 and stimulate its GTPase activity. This GAP activity toward Rab5, however, cannot be extended to other Rab GTPases such as Rab3, Rab4, and Rab6, indicating that it is not a generic GAP for the Rab family of GTPases that regulate intracellular membrane fusion during endocytosis and exocytosis. The findings indicate a level of structural similarity between Ras and Rab5 unexpected from their primary sequences. They also suggest a possible signal transduction regulation of the Rab5-dependent endosome fusion via the Ras GAP.

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity.

The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function.

VPS9 domains can act as guanosine nucleotide exchange factors (GEFs) against small G proteins of the Rab5 family. Saccharomyces cerevisiae vps9Δ mutants have trafficking defects considerably less severe than multiple deletions of the three cognate Rab5 paralogs (Vps21, Ypt52, and Ypt53). Here, we show that Muk1, which also contains a VPS9 domain, acts as a second GEF against Vps21, Ypt52, and Ypt53. Muk1 is partially redundant with Vps9 in vivo, with vps9Δ muk1Δ double mutant cells displaying hypersensitivity to temperature and ionic stress, as well as profound impairments in endocytic and Golgi endosome trafficking, including defects in sorting through the multivesicular body. Cells lacking both Vps9 and Muk1 closely phenocopy double and triple knock-out strains lacking Rab5 paralogs. Microscopy and overexpression experiments demonstrate that Vps9 and Muk1 have distinct localization determinants. These experiments establish Muk1 as the second Rab5 GEF in budding yeast.

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.

Although worldwide concerns have emerged about environmental factors that display carcinogenic and reprotoxic effects, little is known about the mechanism(s) by which these chemicals alter testicular function. Using the 42GPA9 Sertoli cell line, we recently reported that one widely used lipid-soluble pesticide, Lindane impairs gap junctional intercellular communication by promoting the intracellular localization of Connexin 43 (Cx43), a tumor suppressor. We showed here that this chemical triggered the accumulation of Cx43 within Rab5 positive endosomes. Interestingly, evidence is provided that Lindane-induced Cx43 endocytosis did not stem on alteration of Cx43 partition in lipid rafts. Lindane induced concomitantly Cx43 phosphorylation and activation of extracellular signal-regulated kinases (ERK) but not of JNK and p38 mitogen- activated protein kinases. Inhibition of ERK pathway by PD98059, a MEK1-specific inhibitor, prevented Lindane-induced Cx43 phosphorylation, restored Cx43 membranous localization and gap junction coupling. Altogether, these findings provide the first evidence that Lindane-altered Cx43 endocytosis requires ERK activation. Such inappropriate activation of the mitogenic MAPK pathway and inactivation of the tumor suppressor Cx43 by Lindane may participate in the promotion of neoplastic cell growth.

Rin1, the prototype of a new family of multidomain Rab5 exchange factors, has been shown to play an important role in the endocytosis of the epidermal growth factor receptor (EGFR). Herein, we examined the role of Rin1 in the down-regulation of EGFR following EGF stimulation. We observed that overexpression of Rin1 accelerates EGFR degradation in EGF-stimulated cells. In concordance, depletion of endogenous Rin1 by RNA interference resulted in a substantial reduction of EGFR degradation. We showed that Rin1 interacts with signal-transducing adaptor molecule 2 (STAM2), a protein that associates with hepatocyte growth factor-regulated substrate and plays a key role in the endosomal sorting machinery. Green fluorescent protein (GFP)-Rin1 co-localizes with hemagglutinin (HA)-STAM2 and with endogenous hepatocyte growth factor-regulated substrate. Furthermore, wild type STAM2, but not a deletion mutant lacking the SH3 domain, co-immunoprecipitates with endogenous Rin1. This interaction is dependent on the proline-rich domain (PRD) of Rin1 as Rin1DeltaPRD, a mutant lacking the PRD, does not interact with STAM2. Moreover, EGFR degradation was not accelerated by expression of the Rin1DeltaPRD mutant. Together these results suggest that Rin1 regulates EGFR degradation in cooperation with STAM, defining a novel role for Rin1 in regulating endosomal trafficking.

BACKGROUND

Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing.

RESULTS

We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third.

CONCLUSIONS

These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production.