The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway.

rab4 is a ras-like GTP-binding protein that associates with early endosomes in a cell cycle-dependent fashion. To determine its role during endocytosis, we generated stable cell lines that overexpressed mutant or wild-type rab4. By measuring endocytosis, transport to lysosomes, and recycling, we found that overexpression of wild-type rab4 had differential effects on the endocytic pathway. Although initial rates of internalization and degradation were not inhibited, the transfectants exhibited a 3-fold decrease in fluid phase endocytosis as well as an alteration in transferrin receptor (Tfn-R) recycling. Wild-type rab4 caused a redistribution of Tfn-R's from endosomes to the plasma membrane. It also blocked iron discharge by preventing the delivery of Tfn to acidic early endosomes, instead causing Tfn accumulation in a population of nonacidic vesicles and tubules. rab4 thus appears to control the function or formation of endosomes involved in recycling.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading

BACKGROUND: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. RESULTS: The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. CONCLUSIONS: We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.     

Regulation of membrane transport through the endocytic pathway by rabGTPases.

Small GTP binding proteins of the rab family are associated with the cytoplasmic surface of compartments of the central vacuolar system. Several of them, including rab5, rab4 and rab11, are localized to early endocytic organelles where they regulate distinct events in the transferrin receptor pathway. Whereas rab5 is controlling transport to early endosomes, rab4 and rab11 are involved in the regulation of recycling back to the plasma membrane. How GTP-hydrolysis of rab bound GTP is related to the role of these proteins in endocytosis is not yet known, but quick progress is being made towards this goal through the identification of proteins regulating the activity of these rab proteins.     

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.     

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.

     

Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.     

Cholesterol controls lipid endocytosis through Rab11

Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.     

Rab 7: an important regulator of late endocytic membrane traffic

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.     

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

rab4 regulates transport to the apical plasma membrane in Madin-Darby canine kidney cells.

The small GTPase rab4 is associated with early endosomes and regulates membrane recycling in fibroblasts. rab4 is present in epithelial cells; however, neither its localization nor function has been established in this cell type. We transfected Madin-Darby canine kidney cells with rab4, the GTPase-deficient mutant rab4Q67L, and the dominant negative mutant rab4S22N that poorly binds guanine nucleotides. Confocal immunofluorescence microscopy showed that rab4 was concentrated on internal structures at the lateral side of the cell around the nucleus. Quantitative immunoelectron microscopy revealed that the majority of rab4 was localized in the upper third of the cytoplasm. In cell surface binding experiments with (125)I-transferrin, we found a redistribution of transferrin receptor from the basolateral to the apical plasma membrane in cells expressing rab4 and rab4Q67L. After accumulation of transferrin at 16 degrees C in basolateral early endosomes, rab4 and rab4Q67L increased the amount of apically targeted transferrin receptor. A qualitatively similar effect was obtained in control cells treated with brefeldin A. The effects of brefeldin A and rab4 on apical targeting of transferrin receptor were not additive, suggesting that brefeldin A and rab4 may act in the same transport pathway from common endosomes.     

Clathrin hub expression affects early endosome distribution with minimal impact on receptor sorting and recycling

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.     

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.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.     

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.     

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.     

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)-RME-6 fusion protein primarily localizes to clathrin-coated pits, physically interacts with alpha-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants, transport from the plasma membrane to endosomes is defective, and small 110-nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin-coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.     

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.     

Visualization of receptor-mediated endocytosis in yeast

We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.