Regulation of endosome fusion.

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.     

The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization

Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural features in EEA1 required for binding to these ligands. We have found that the FYVE domain is critical for both PtdIns3P and Rab5 binding. Whereas PtdIns3P binding only required the FYVE domain, Rab5 binding additionally required a 30-amino acid region directly adjacent to the FYVE domain. Microinjection of glutathione S-transferase fusion constructs into Cos cells revealed that the FYVE domain alone is insufficient for localization to cellular membranes; the upstream 30-amino acid region required for Rab5 binding must also be present for endosomal binding. The importance of Rab5 in membrane binding of EEA1 is underscored by the finding that the increased expression of wild-type Rab5 increases endosomal binding of EEA1 and decreases its dependence on PtdIns3P. Thus, the levels of Rab5 are rate-limiting for the recruitment of EEA1 to endosome membranes. PtdIns3P may play a role in modulating the Rab5 EEA1 interaction.     

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3‐phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain‐containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid‐phase marker dextran were retained in abnormally small endosomes in Phafin2‐depleted cells. In yeast two‐hybrid analysis we identified Phafin2 as a novel interactor of the endosomal‐tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid‐phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.     

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.     

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P₂, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.David J. Gillooly and Camilla Raiborg contributed equally     

A PtdIns(3)P-specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells.

Salmonella invade nonphagocytic cells by eliciting their own internalization; upon contact with the host cell, the bacteria induce membrane ruffles highly localized to the point of contact between the invading bacterium and the host cell. The bacterium is then internalized into an unusual cytosolic organelle, the Salmonella-containing vacuole (SCV). Early endosomal markers (including EEA1) have recently been shown to be associated with the SCV shortly after invasion. EEA1, a protein involved in early endosome fusion, is recruited to early endosomal membranes in part by the interaction between its FYVE finger and phosphatidylinositol 3-phosphate [PtdIns(3)P], a characteristic lipid of early endosomes. This suggests a possible role for PtdIns(3)P during Salmonella infection. To investigate this, we generated a highly specific probe for PtdIns(3)P that was used to follow invasion of Salmonella in nonphagocytic cells. Here, we show that PtdIns(3)P is present on the membranes of SCVs shortly after invasion and also that it is present on the membrane ruffles produced immediately prior to invasion. We also show that this specific probe cycles on and off the membranes of nascent SCVs even when PtdIns 3-kinase activity is inhibited, demonstrating that invading Salmonella influence the composition of the membranes that envelop them during invasion.     

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm² in the search for a second endosome with which to interact.     

Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.     

Role of Rab5 in the recruitment of hVps34/p150 to the early endosome

PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo . We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.     

Phosphatidylinositol 3-phosphate recognition by the FYVE domain.

Recognition of phosphatidylinositol 3-phosphate (Ptdlns(3)P) is crucial for a broad range of cellular signaling and membrane trafficking events regulated by phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion, involves two beta hairpins and an alpha helix. Specific amino acids, including those of the FYVE domain's conserved RRHHCRQCGNIF motif, contact soluble and micelle-embedded lipid and provide specificity for Ptdlns(3)P over Ptdlns(5)P and Ptdlns, as shown by heteronuclear magnetic resonance spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by ovel structural features and its ptdlns(3)P-binding site.     

Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.     

Ca2+ and N-ethylmaleimide-sensitive factor differentially regulate disassembly of SNARE complexes on early endosomes

The endosome-associated protein Hrs inhibits the homotypic fusion of early endosomes. A helical region of Hrs containing a Q-SNARE motif mediates this effect as well as its endosomal membrane association via SNAP-25, an endosomal receptor for Hrs. Hrs inhibits formation of an early endosomal SNARE complex by displacing VAMP-2 from the complex, suggesting a mechanism by which Hrs inhibits early endosome fusion. We examined the regulation of endosomal SNARE complexes to probe how Hrs may function as a negative regulator. We show that although NSF dissociates the VAMP-2.SNAP-25.syntaxin 13 complex, it has no effect on the Hrs-containing complex. Whereas Ca(2+) dissociates the Hrs-containing complex but not the VAMP-2-containing SNARE complex. This is the first demonstration of differential regulation of R/Q-SNARE and all Q-SNARE-containing SNARE complexes. Ca(2+) also reverses the Hrs-induced inhibition of early endosome fusion in a tetanus toxin-sensitive manner and removes Hrs from early endosomal membranes. Moreover, Hrs inhibition of endosome fusion and its endosomal localization are sensitive to bafilomycin, implying a role for luminal Ca(2+). Thus, Hrs may bind a SNARE protein on early endosomal membranes negatively regulating trans-SNARE pairing and endosomal fusion. The release of Ca(2+) from the endosome lumen dissociates Hrs, allowing a VAMP-2-containing complex to form enabling fusion.     

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.     

Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes

Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.     

FYVE domain targets Pib1p ubiquitin ligase to endosome and vacuolar membranes

Signaling by phosphatidylinositol 3-kinases (PI3Ks) is often mediated by proteins which bind PI3K products directly and are localized to intracellular membranes rich in PI3K products. The FYVE finger domain binds with high specificity to PtdIns3P and proteins containing this domain have been shown to be important components of diverse PI3K signaling pathways. The genome of the yeast Saccharomyces cerevisiae encodes five proteins containing FYVE domains, including Pib1p, whose function is unknown. In addition to a FYVE finger motif, the primary structure of Pib1p contains a region rich in cysteine and histidine residues that we demonstrate binds 2 mol eq of zinc, consistent with this region containing a RING structural domain. The Pib1p RING domain exhibited E2-dependent ubiquitin ligase activity in vitro, indicating that Pib1p is an E3 RING-type ubiquitin ligase. Fluorescence microscopy was used to demonstrate that a GFP-Pib1p fusion protein localized to endosomal and vacuolar membranes and deletional analysis of Pib1p domains indicated that localization of GFP-Pib1p is mediated solely by the FYVE domain. These results suggest that Pib1p mediates ubiquitination of a subset of cellular proteins localized to endosome and vacuolar membranes, and they expand the repertoire of PI3K-regulated pathways identified in eukaryotic cells.     

Dissociation of RalA from synaptic membranes by Ca2+/calmodulin.

Ras-related small GTP-binding proteins execute many cellular functions, such as cell growth, differentiation, cytoskeletal reorganization, membrane trafficking, and membrane fusion. RalA belongs to the superfamily of Ras-related small GTP-binding proteins. Synaptic vesicles (SV) contain small GTP-binding proteins, where RalA, Rab3A, and Rab5A are the major GTP-binding proteins. It has been postulated that a cycling of these proteins between membrane-bound and soluble states is required for regulating cellular functions. Calmodulin (CaM) was found to dissociate Rab3A from SV membranes by forming a 1:1 complex with Ca2+/CaM. RalA was also found to be a Ca2+/CaM-binding protein. Therefore, we examined if Ca2+/CaM can also cause the RalA to dissociate from SV membranes. In this study, we identified that Ca2+/CaM dissociates RalA as well as Rab3A from synaptic vesicles.     

The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.     

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.     

Cell-cycle-dependent binding kinetics for the early endosomal tethering factor EEA1

Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein-tagged EEA1 (EEA1-GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half-life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long-lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane-binding characteristics. This might be a general mechanism to fine-tune and control tethering and fusion of early endosomes.