Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.     

Expression of dominant negative rab5 in HeLa cells regulates endocytic trafficking distal from the plasma membrane

Ligand-mediated endocytosis is an important regulatory mechanism of epidermal growth factor (EGF) receptor (EGFR) signal transduction. Coordinated EGFR internalization and degradation function to regulate the spatial and temporal components of EGFR-effector interactions. In an effort to better understand the molecular mechanisms that control these events, we examined the role of rab5 in the endocytic trafficking of the EGFR. Rab5 is a 25-kDa guanine nucleotide binding protein that has previously been shown to be involved in the early stages of endocytic trafficking. Using adenovirally expressed dominant negative and constitutively active rab5 [rab5(S34N) and rab5(Q79L)] in cells with endogenous EGFRs, we have found that the guanine nucleotide binding state of rab5 has no bearing on the rate of EGFR endocytosis. However, expression of dominant negative rab5 affects downstream endocytic trafficking by slowing the ligand-induced disappearance of total cellular EGFR. Using confocal microscopy to examine EGF/EGFR and rab5 localization indicates that the activity of rab5 governs whether internalized EGF/EGFR and rab5 co-localize. Transferrin, which internalizes via a constitutively internalized cell surface receptor, co-sediments with rab5(WT), but not rab5(S34N) on sucrose gradients. Taken together, these data are consistent with rab5 functioning to regulate intracellular endocytic trafficking distal from the plasma membrane.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.     

Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.     

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases

The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment.     

Receptor and membrane recycling can occur with unaltered efficiency despite dramatic Rab5(q79l)-induced changes in endosome geometry

Current models for sorting in the endosomal compartment suggest that endosomal geometry plays a significant role as membrane-bound proteins accumulate in tubular regions for recycling, and lumenal markers accumulate in large vacuolar portions for delivery to lysosomes. Rab5, a small molecular weight GTPase, functions in the formation and maintenance of the early/sorting endosome. Overexpression of the constitutively active form, Rab5(Q79L), leads to enhanced endosome fusion resulting in the enlargement of early endosomes. Using an adenoviral expression system to regulate the time and level of Rab5(Q79L) overexpression in HeLa cells, we find that although endosomes are dramatically enlarged, the rates of transferrin receptor-mediated endocytosis and recycling are unaffected. Moreover, despite the enlarged endosome phenotype, neither the rate of internalization of a fluid phase marker nor the rate of recycling of a bulk lipid marker were affected. These results suggest that GTP hydrolysis by Rab5 is rate-limiting for endosome fusion but not for endocytic trafficking and that early endosome geometry may be a less critical determinant of sorting efficiencies than previously thought.     

Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5

The small GTPase Rab5 controls the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. Expression of a GTPase-defective mutant, Rab5(Q79L), is known to cause formation of enlarged early endosomes. The ability of Rab5-GTP to recruit multiple effectors raises the question whether the Rab5(Q79L)-induced giant endosomes simply represent enlarged early endosomes or whether they have a more complex phenotype. In this report, we have addressed this issue by generating a HEp2 cell line with inducible expression of Rab5(Q79L) and performing ultrastructural analysis of Rab5(Q79L)-induced endosomes. We find that Rab5(Q79L) not only induces formation of enlarged early endosomes but also causes enlargement of later endocytic profiles. Most strikingly, Rab5(Q79L) causes formation of enlarged multivesicular endosomes with a large number of intraluminal vesicles, and endosomes that contain both early and late endocytic markers are frequently observed. In addition, we observe defects in the sorting of the EGF receptor and the transferrin receptor through this compartment.     

Human RME-8 is involved in membrane trafficking through early endosomes

RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.     

Rab15 mediates an early endocytic event in Chinese hamster ovary cells.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.     

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

Dual function of rhoD in vesicular movement and cell motility

The trafficking of intracellular membranes requires the coordination of membrane-cytoskeletal interactions. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions. We have previously identified a rho protein, rhoD, which is localized to the plasma membrane and early endosomes. When overexpressed, rhoD alters the actin cytoskeleton and plays an important role in endosome organization. We found that a rhoD mutant exerts its effect on early endosome dynamics through an inhibition in organelle motility. In these studies, the effect of rhoD on endosome dynamics was evaluated in the presence of a constitutively active, GTPase-deficient mutant of rab5, rab5Q79L. As rab5Q79L itself stimulates endosome motility, rhoD might counteract this stimulation, without itself exerting any effect in the absence of rab5 activation. We have now addressed this issue by investigating the effect of rhoD in the absence of co-expressed rab5. We find that rhoDG26V alone alters vesicular dynamics. Vesicular movement, in particular the endocytic/recycling circuit, is altered during processes such as cell motility. Due to the participation of vesicular motility and cytoskeletal rearrangements in cell movement and the involvement of rhoD in both, we have addressed the role of rhoD in this process and have found that rhoDG26V inhibits endothelial cell motility.     

ATP depletion inhibits the endocytosis of ClC-2

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel.     

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella‐containing vacuole

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella‐containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3‐phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30–60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella‐induced filaments (Sifs) was altered by SNX3 knock‐down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.     

An internalization signal in ClC-5, an endosomal Cl-channel mutated in dent's disease

The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent's disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na(+) channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxyl-terminal internalization signal with ubiquitin-protein ligases containing WW domains.     

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

We evaluated the contribution ofrab5a and rab11a to trafficking and signaling of protease-activatedreceptor 2 (PAR2), a receptor for trypsin and tryptase. Agonistsstimulated internalization of PAR2 into early endosomes containingrab5a. Dominant negative rab5aS34N disrupted early endosomes andinhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 wassorted to lysosomes, and rab5a remained in early endosomes. Rab5apromoted and rab5aS34N impeded resensitization of trypsin-inducedcalcium mobilization. Rab11a was detected in the Golgi apparatus withPAR2, and PAR2 agonists stimulated redistribution of rab11a intovesicles containing PAR2 that migrated to the cell surface. Dominantnegative rab11aS25N was mostly confined to the Golgi apparatus.Although expression of rab11aS25N caused retention of PAR2 in the Golgiapparatus, it did not abolish trafficking of PAR2 to the cell surface.However, expression of wild-type rab11a accelerated both recovery ofPAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus tothe plasma membrane.

     

Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.     

Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes

Adiponectin and leptin are two adipokines secreted by white adipose tissue that regulate insulin sensitivity. Previously we reported that adiponectin but not leptin release depends on GGA-coated vesicle formation, suggesting that leptin and adiponectin may follow different secretory routes. Here we have examined the intracellular trafficking pathways that lead to the secretion of these two hormones. While adiponectin and leptin displayed distinct localization in the steady-state, treatment of adipocytes with brefeldin A inhibited both adiponectin and leptin secretion to a similar level, indicating a common requirement for class III ADP-ribosylating factors and an intact Golgi apparatus. Adiponectin secretion was significantly reduced by endosomal inactivation in both 3T3L1 and rat isolated adipocytes, whereas this treatment had no effect on leptin secretion. Importantly, endosomal inactivation completely abolished the insulin stimulatory effect on adiponectin release in rat adipocytes. Confocal microscopy studies revealed colocalization of adiponectin with endogenous rab11 a marker for the recycling endosome, and with expressed rab5-GFP mutant (rab5Q75L) a marker for the early endosome compartment. Colocalization of adiponectin and rab5Q75L was increased in endosome inactivated cells. Consistent with these findings adiponectin secretion was reduced in cells expressing mutants of Rab11 and Rab5 proteins. In contrast, expression of an inactive (kinase dead) mutant of Protein Kinase D1 moderately but significantly inhibited leptin secretion without altering adiponectin secretion. Taken together, these results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and that endosomal compartments are required for adiponectin but not for leptin secretion.     

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.     

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.     

The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.