G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
early endosome antigen 1
G00000609 (Mus musculus)

Databases (7)

ENSG00000102189 (Ensembl human gene)
8411 (Entrez Gene)
1009 (G2Cdb plasticity & disease)
EEA1 (GeneCards)
605070 (OMIM)
Marker Symbol
HGNC:3185 (HGNC)
Protein Sequence
Q15075 (UniProt)

Synonyms (1)

  • ZFYVE2

Literature (37)

Pubmed - other

  • Identification of c-Cbl as a new ligase for insulin-like growth factor-I receptor with distinct roles from Mdm2 in receptor ubiquitination and endocytosis.

    Sehat B, Andersson S, Girnita L and Larsson O

    Department of Oncology and Pathology, Karolinska Institutet, Cancer Center Karolinska, Karolinska University Hospital, Stockholm, Sweden.

    The insulin-like growth factor receptor (IGF-IR) plays several pivotal roles in cancer. Although most studies on the function of the IGF-IR have been attributed to kinase-dependent signaling, recent findings by our group and others have implicated biological roles mediated by ubiquitination of the receptor. As previously reported, the E3 ligases Mdm2 and Nedd4 mediate IGF-IR ubiquitination. Here we show that c-Cbl is a novel E3 ligase for IGF-IR. On ligand stimulation, both Mdm2 and c-Cbl associate with IGF-IR and mediate receptor polyubiquitination. Whereas Mdm2 catalyzed lysine 63 (K63) chain ubiquitination, c-Cbl modified IGF-IR through K48 chains. Mdm2-mediated ubiquitination occurred when cells were stimulated with a low concentration (5 ng/mL) of IGF-I, whereas c-Cbl required high concentrations (50-100 ng/mL). Mdm2-ubiquitinated IGF-IR was internalized through the clathrin endocytic pathway whereas c-Cbl-ubiquitinated receptors were endocytosed via the caveolin route. Taken together, our results show that c-Cbl constitutes a new ligase responsible for the ubiquitination of IGF-IR and that it complements the action of Mdm2 on ubiquitin lysine residue specificity, responsiveness to IGF-I, and type of endocytic pathway used. The actions and interactions of Mdm2 and c-Cbl in the ubiquitination and endocytosis of IGF-IR may have implications in cancer. In addition, identification and functional characterization of new E3 ligases are important in itself because therapeutic targeting of substrate-specific E3 ligases is likely to represent a critical strategy in future cancer treatment.

    Cancer research 2008;68;14;5669-77

  • Human autoantibodies against early endosome antigen-1 enhance excitatory synaptic transmission.

    Selak S, Paternain AV, Fritzler MJ and Lerma J

    Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Aptdo 18, 03550 Sant Joan d'Alacant, Spain.

    Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl-D-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies.

    Neuroscience 2006;143;4;953-64

  • Palmitoyl protein thioesterase 1 (PPT1) deficiency causes endocytic defects connected to abnormal saposin processing.

    Ahtiainen L, Luiro K, Kauppi M, Tyynelä J, Kopra O and Jalanko A

    National Public Health Institute, Department of Molecular Medicine, Biomedicum Helsinki, P.O. Box 104, 00251 Helsinki, Finland.

    Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.

    Experimental cell research 2006;312;9;1540-53

  • Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1.

    Cabezas A, Pattni K and Stenmark H

    Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    Yeast Fab1 is a phosphatidylinositol 3-phosphate 5-kinase involved in endocytic membrane traffic and vacuole homeostasis. Here we have cloned and sequenced the cDNA for the human homologue of Fab1, PIKfyve. The cDNA has an open reading frame of 6294 bp and encodes a 2098-amino acid protein with a calculated molecular mass of 237 kDa, containing a phosphatidylinositol 3-phosphate-binding FYVE domain, a DEP domain, a chaperonin-like domain, and a phosphoinositide kinase domain. The human genome contains a single PIKfyve gene, which comprises 38 exons on chromosomal locus 2q34. PIKfyve is expressed as a single molecular species in a number of human cell lines derived from different tissues. The exogenously expressed protein was found to localize mainly to early endosomes containing two other FYVE domain proteins, EEA1 and Hrs. The endosomal membrane localization of PIKfyve was studied in more detail by examining cells transfected with a constitutively active mutant of the small GTPase Rab5, whose expression results in the enlargement of early endosomes. We show that PIKfyve is distributed in microdomains that are distinct from those occupied by EEA1 and Hrs.

    Gene 2006;371;1;34-41

  • Effect of 3-methyladenine on the fusion process of macropinosomes in EGF-stimulated A431 cells.

    Araki N, Hamasaki M, Egami Y and Hatae T

    Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan. naraki@med.kagawa-u.ac.jp

    In the process of receptor-mediated endocytosis, the fusion of endosomes in vitro is known to be inhibited by wortmannin or LY294002; inhibitors of phosphoinositide 3-kinase (PI3K), suggesting that the activity of PI3K is required for the fusion of early endosomes. In macropinocytosis, a process of bulk fluid-phase endocytosis, however, it remains unclear whether PI3K is required for the fusion of macropinosomes, since the macropinosome formation is inhibited by the PI3K inhibitors. In this study, we examined the effect of 3-methlyadenine (3-MA), which shows a distinct specificity to the PI3K classes from wortmannin and LY294002, on the macropinosome formation and fusion in EGF-stimulated A431 cells. Unlike wortmannin or LY294002, 3-MA did not inhibit the uptake of fluorescent dextran by macropinocytosis. However, the fusion of macropinosomes was inhibited by 3-MA. By imaging of live-cells expressing fluorescent protein-fused tandem FYVE domains, we found that PtdIns(3)P appeared on the macropinosomal membrane shortly after the closure of macropinocytic cups and remained on macropinosomes even at 60-min age. The production of PtdIns(3)P and the recruitment of EEA1 to macropinosomes were abolished by the 3-MA treatment. Therefore, it is likely that 3-MA impairs recruitment of EEA1 by inhibiting PtdIns(3)P production and resultantly blocks the fusion of macropinosomes. These results suggest that the local production of PtdIns(3)P implicates the fusion of macropinosomes via EEA1 as well as conventional early endosomes. However, the long association of PtdIns(3)P with macropinosomes may well be a cell-type specific feature of A431 cells.

    Cell structure and function 2006;31;2;145-57

  • Investigation of the binding geometry of a peripheral membrane protein.

    Brunecky R, Lee S, Rzepecki PW, Overduin M, Prestwich GD, Kutateladze AG and Kutateladze TG

    Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.

    A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.

    Funded by: Biotechnology and Biological Sciences Research Council: BB_BBS/B/10714; NCI NIH HHS: CA085716, R01 CA085716, R01 CA085716-05; NIGMS NIH HHS: GM067655, GM089074, R01 GM067655; NINDS NIH HHS: NS29632, R01 NS029632; Wellcome Trust: WT071684

    Biochemistry 2005;44;49;16064-71

  • Phosphorylation of EEA1 by p38 MAP kinase regulates mu opioid receptor endocytosis.

    Macé G, Miaczynska M, Zerial M and Nebreda AR

    European Molecular Biology Laboratory, Heidelberg, Germany.

    Morphine analgesic properties and side effects such as tolerance are mediated by the mu opioid receptor (MOR) whose endocytosis is considered of primary importance for opioid pharmacological effects. Here, we show that p38 mitogen-activated protein kinase (MAPK) activation is required for MOR endocytosis and sufficient to trigger its constitutive internalization in the absence of agonist. Further studies established a functional link between p38 MAPK and the small GTPase Rab5, a key regulator of endocytosis. Expression of an activated mutant of Rab5 stimulated endocytosis of MOR ligand-independently in wild-type but not in p38alpha-/- cells. We found that p38alpha can phosphorylate the Rab5 effectors EEA1 and Rabenosyn-5 on Thr-1392 and Ser-215, respectively, and these phosphorylation events regulate the recruitment of EEA1 and Rabenosyn-5 to membranes. Moreover, phosphomimetic mutation of Thr-1392 in EEA1 can bypass the requirement for p38alpha in MOR endocytosis. Our results highlight a novel mechanism whereby p38 MAPK regulates receptor endocytosis under physiological conditions via phosphorylation of Rab5 effectors.

    The EMBO journal 2005;24;18;3235-46

  • Transferrin receptor co-localizes and interacts with the hemochromatosis factor (HFE) and the divalent metal transporter-1 (DMT1) in trophoblast cells.

    Gruper Y, Bar J, Bacharach E and Ehrlich R

    Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.

    Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. The placenta is the entry site for iron, which is delivered to the developing embryo. Iron is taken up by syncytiotrophoblast cells and is transported unidirectionally from mother to fetus against a concentration gradient. Several iron transporters and regulators were recently characterized, including DMT1 and ferroportin/Ireg1 that transport iron through membranes, and HFE that regulates TfR-mediated iron uptake. In this study we demonstrate that in a differentiated choriocarcinoma cell line BeWo, HFE, and TfR are localized mainly in recycling endosomes and a small percentage of these complexes is observed in late endosomes with DMT1 while in trophoblast cells, the level of TfR is significantly lower and it is detected with HFE and DMT1 mainly in late endosomes. Most interestingly, TfR and HFE, as well as TfR and DMT1 interact in placental trophoblast cells. Based on previous and these data we suggest that the level of intracellular iron may regulate both TfR expression (on the post-transcriptional and the post-translational levels) and TfR trafficking/transcytosis in polarized cells.

    Journal of cellular physiology 2005;204;3;901-12

  • Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron.

    Johnson DM, Yamaji S, Tennant J, Srai SK and Sharp PA

    School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK.

    A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.

    FEBS letters 2005;579;9;1923-9

  • Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells.

    Galperin E, Verkhusha VV and Sorkin A

    Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.

    Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

    Nature methods 2004;1;3;209-17

  • Characterization of early endosome antigen 1 in neural tissues.

    Selak S, Braun JE and Fritzler MJ

    Department of Neural Plasticity, Cajal Institute, Madrid, Spain.

    The finding that patients and mice bearing autoantibodies directed against early endosome antigen 1 (EEA1) develop neurological signs and deficits prompted an investigation of EEA1 distribution, localization, and interaction with synaptic proteins found in neural tissues. We detected EEA1 in a variety of neural tissues and in cells of neural origin where it co-localized with SNAP-25. The interaction between EEA1 and SNAP-25 was dependent on the leucine zipper and a newly identified methyl-accepting domain of EEA1. The C-terminal zinc-binding FYVE finger motif (EEA1(1271-1411)) of EEA1 also interacted with native SNAP-25 but only in the presence of 100microM Ca(2+). In contrast, EEA1 did not bind to cysteine string protein or synapsin in these binding assays. These results suggest that EEA1 is involved in neuronal synaptic vesicle function and axonal transport and growth. EEA1 may undergo calcium-dependent conformational changes that are required for binding to SNAP-25.

    Biochemical and biophysical research communications 2004;323;4;1334-42

  • Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment.

    Coumailleau F, Das V, Alcover A, Raposo G, Vandormael-Pournin S, Le Bras S, Baldacci P, Dautry-Varsat A, Babinet C and Cohen-Tannoudji M

    Unité Biologie du Développement, CNRS URA 2578, Institut Pasteur, 75724 Paris Cedex 15, France.

    Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

    Molecular biology of the cell 2004;15;10;4444-56

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Association of early endosomal autoantigen 1 with macropinocytosis in EGF-stimulated A431 cells.

    Hamasaki M, Araki N and Hatae T

    Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, Japan.

    Association of early endosomal autoantigen 1 (EEA1) with macropinosomes was examined in EGF-stimulated A431 cells by dual labeling with immunofluorescence of anti-EEA1 and FITC-dextran (FDx), a fluid-phase endocytic marker. Addition of EGF to A431 cells drastically enhanced macropinosome formation. Newly formed macropinosomes labeled with 5-min pulse of FDx were located at the cell periphery and labeled weakly for EEA1. After a 5-min chase, these macropinosomes aggregated and frequently fused with each other. Immunofluorescence showed that EEA1 appeared on the membrane of FDx-labeled macropinosomes at that time, suggesting that EEA1 functioned in homotypic macropinosome fusion. With longer chase (30-60 min), macropinosomes decreased in number and size, indicating that FDx was largely exocytosed via recycling compartments. A small amount of FDx-labeled macropinosomes remained in the perinuclear region even at 60 min after pulse labeling. They were EEA1-positive but negative for cathepsin D, a lysosomal enzyme. This indicates that macropinosomes do not mature to late endosomes or fuse with lysosomes. Instead, EEA1 continuously mediates homotypic fusion as long as the macropinosomes persist.

    The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 2004;277;2;298-306

  • Endofin recruits TOM1 to endosomes.

    Seet LF, Liu N, Hanson BJ and Hong W

    Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore. mcbslf@imcb.a-star.edu.sg

    Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.

    The Journal of biological chemistry 2004;279;6;4670-9

  • Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis.

    Galperin E and Sorkin A

    Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

    Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.

    Funded by: NCI NIH HHS: CA089151

    Journal of cell science 2003;116;Pt 23;4799-810

  • Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes.

    Fratti RA, Chua J and Deretic V

    Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131, USA.

    Mycobacterium tuberculosis survives in the infected host by parasitizing macrophages in which the bacillus resides in a specialized phagosome sequestered from the phagolysosomal degradative pathway. Here we report a role of the stress-induced p38 mitogen-activated protein kinase (p38 MAPK) in the component of M. tuberculosis phagosome maturation arrest that has been linked previously to the reduced recruitment of the endosomal and phagosomal membrane-tethering molecule called early endosome autoantigen 1 (EEA1; Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S., and Deretic, V. (2001) J. Cell Biol. 154, 631-644). A pharmacological inhibition of M. tuberculosis var. bovis Bacillus Calmette-Guérin-induced p38 MAPK activity caused a marked increase in EEA1 colocalization with mycobacterial phagosomes. Consistent with the increase in EEA1 association and its role in phagosomal maturation, the pharmacological block of p38 activity caused phagosomal acidification and enrichment of the late endocytic markers lysobisphosphatidic acid and CD63 (lysosomal integral membrane protein 1) on mycobacterial phagosomes. A negative regulatory role of p38 MAPK activation in phagosome maturation was further demonstrated by converse experiments with latex bead phagosomes. Artificial activation of p38 MAPK caused a decrease in EEA1 colocalization with model latex bead phagosomes, which normally acquire EEA1 and subsequently mature into the phagolysosome. These findings show that p38 MAPK activity contributes to the arrest of M. tuberculosis phagosome maturation and demonstrate a negative regulatory role of p38 in phagolysosome biogenesis.

    Funded by: NIAID NIH HHS: AI 45148

    The Journal of biological chemistry 2003;278;47;46961-7

  • Identification of a novel domain in two mammalian inositol-polyphosphate 5-phosphatases that mediates membrane ruffle localization. The inositol 5-phosphatase skip localizes to the endoplasmic reticulum and translocates to membrane ruffles following epidermal growth factor stimulation.

    Gurung R, Tan A, Ooms LM, McGrath MJ, Huysmans RD, Munday AD, Prescott M, Whisstock JC and Mitchell CA

    Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.

    SKIP (skeletal muscle and kidney enriched inositol phosphatase) is a recently identified phosphatidylinositol 3,4,5-trisphosphate- and phosphatidylinositol 4,5-bisphosphate-specific 5-phosphatase. In this study, we investigated the intracellular localization of SKIP. Indirect immunofluorescence and subcellular fractionation showed that, in serum-starved cells, both endogenous and recombinant SKIP colocalized with markers of the endoplasmic reticulum (ER). Following epidermal growth factor (EGF) stimulation, SKIP transiently translocated to plasma membrane ruffles and colocalized with submembranous actin. Data base searching demonstrated a novel 128-amino acid domain in the C terminus of SKIP, designated SKICH for SKIP carboxyl homology, which is also found in the 107-kDa 5-phosphatase PIPP and in members of the TRAF6-binding protein family. Recombinant SKIP lacking the SKICH domain localized to the ER, but did not translocate to membrane ruffles following EGF stimulation. The SKIP SKICH domain showed perinuclear localization and mediated EGF-stimulated plasma membrane ruffle localization. The SKICH domain of the 5-phosphatase PIPP also mediated plasma membrane ruffle localization. Mutational analysis identified the core sequence within the SKICH domain that mediated constitutive membrane association and C-terminal sequences unique to SKIP that contributed to ER localization. Collectively, these studies demonstrate a novel membrane-targeting domain that serves to recruit SKIP and PIPP to membrane ruffles.

    The Journal of biological chemistry 2003;278;13;11376-85

  • Determinants of Rab5 interaction with the N terminus of early endosome antigen 1.

    Merithew E, Stone C, Eathiraj S and Lambright DG

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01605, USA.

    The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C(2)H(2) Zn(2+) finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C(2)H(2) Zn(2+) finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C(2)H(2) Zn(2+) finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C(2)H(2) Zn(2+) finger play a critical role in the interaction with Rab5. Although the homologous C(2)H(2) Zn(2+) finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C(2)H(2) Zn(2+) finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C(2)H(2) Zn(2+) finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C(2)H(2) Zn(2+) finger of EEA1.

    Funded by: NIGMS NIH HHS: GM56324

    The Journal of biological chemistry 2003;278;10;8494-500

  • Functional expression of CCR2 by human fetal astrocytes.

    Andjelkovic AV, Song L, Dzenko KA, Cong H and Pachter JS

    Blood-Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.

    Astrocytes from different sources bind the chemokine monocyte chemoattractant factor (MCP-1), yet functional expression in these cells of CCR2, the major receptor for this ligand, has been a matter of controversy. Here we show that cultured human fetal astrocytes express CCR2 at the mRNA and protein levels, and display chemotaxis and calcium flux in response to MCP-1. Surface CCR2 protein expression and MCP-1 binding activity were observed to undergo near parallel downmodulation and recovery following MCP-1 exposure, supporting the argument that CCR2, and not another receptor, mediates MCP-1 ligation in these cells. Downmodulation was further determined to occur via receptor internalization, and to apparently proceed via both clathrin-coated vesicles and caveolae, the latter being a novel mode for the endocytosis of chemokine receptors. Insofar as MCP-1 is thought to mediate inflammatory and developmental processes within the central nervous system (CNS), such astrocyte responses to this chemokine are likely to significantly impact physiological and pathophysiological events at the blood-brain barrier and within the CNS parenchyma.

    Funded by: NIMH NIH HHS: R0-1-MH54718

    Journal of neuroscience research 2002;70;2;219-31

  • The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking.

    Kauppi M, Simonsen A, Bremnes B, Vieira A, Callaghan J, Stenmark H and Olkkonen VM

    Department of Molecular Medicine, National Public Health Institute (KTL), Biomedicum, PO Box 104, FIN-00251 Helsinki, Finland.

    Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells, overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex. In BHK cells, the Q64L mutant caused the accumulation of a fluid phase marker, TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers. Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF. These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.

    Journal of cell science 2002;115;Pt 5;899-911

  • Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER.

    Valsdottir R, Hashimoto H, Ashman K, Koda T, Storrie B and Nilsson T

    Cell Biology and Biophysics Programme, EMBL, Heidelberg, Germany.

    The role of rab33b, a Golgi-specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST-rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin-5 and rabex-5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin-5 and rabex-5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.

    FEBS letters 2001;508;2;201-9

  • Multivalent endosome targeting by homodimeric EEA1.

    Dumas JJ, Merithew E, Sudharshan E, Rajamani D, Hayes S, Lawe D, Corvera S and Lambright DG

    Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

    Early endosome autoantigen localization to early endosomes is mediated by a C-terminal region, which includes a calmodulin binding motif, a Rab5 interaction site, and a FYVE domain that selectively binds phosphatidyl inositol 3-phosphate. The crystal structure of the C-terminal region bound to inositol 1,3-bisphosphate reveals an organized, quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal localization in which domain organization, dimerization, and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. A unique mode of membrane engagement deduced from the quaternary structure of the C-terminal region provides insight into the structural basis of endosome tethering.

    Funded by: NIDDK NIH HHS: DK60564

    Molecular cell 2001;8;5;947-58

  • FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes.

    Raiborg C, Bremnes B, Mehlum A, Gillooly DJ, D'Arrigo A, Stang E and Stenmark H

    Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.

    Journal of cell science 2001;114;Pt 12;2255-63

  • Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi.

    Cheung PC, Trinkle-Mulcahy L, Cohen P and Lucocq JM

    MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K. pcheung1@biochem.dundee.ac.uk

    We have identified a novel protein of predicted molecular mass 40 kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain.

    The Biochemical journal 2001;355;Pt 1;113-21

  • Structural mechanism of endosome docking by the FYVE domain.

    Kutateladze T and Overduin M

    Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA. tatiana.kutateladze@uchsc.edu

    The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.

    Funded by: NCI NIH HHS: CA85716

    Science (New York, N.Y.) 2001;291;5509;1793-6

  • ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6.

    Someya A, Sata M, Takeda K, Pacheco-Rodriguez G, Ferrans VJ, Moss J and Vaughan M

    Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. someyaa@nih.gov

    A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;5;2413-8

  • Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues.

    Gaullier JM, Ronning E, Gillooly DJ and Stenmark H

    Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn(2+)-dependent, and Zn(2+) could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K(D) of about 50 nm and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn(2+) coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.

    The Journal of biological chemistry 2000;275;32;24595-600

  • Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion.

    Allan BB, Moyer BD and Balch WE

    Departments of Cell and Molecular Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    The guanosine triphosphatase Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through interaction with effector molecules, but the molecular mechanisms by which this occurs are unknown. Here, the tethering factor p115 was shown to be a Rab1 effector that binds directly to activated Rab1. Rab1 recruited p115 to coat protein complex II (COPII) vesicles during budding from the endoplasmic reticulum, where it interacted with a select set of COPII vesicle-associated SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) to form a cis-SNARE complex that promotes targeting to the Golgi apparatus. We propose that Rab1-regulated assembly of functional effector-SNARE complexes defines a conserved molecular mechanism to coordinate recognition between subcellular compartments.

    Funded by: NCI NIH HHS: CA58689; NIGMS NIH HHS: GM 33301, GM42336

    Science (New York, N.Y.) 2000;289;5478;444-8

  • The Rab5 effector EEA1 interacts directly with syntaxin-6.

    Simonsen A, Gaullier JM, D'Arrigo A and Stenmark H

    Department of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    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.

    The Journal of biological chemistry 1999;274;41;28857-60

  • Direct interaction of EEA1 with Rab5b.

    Callaghan J, Nixon S, Bucci C, Toh BH and Stenmark H

    Department of Biochemistry, The Norwegian Radium Hospital, Oslo.

    The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion. It binds to endosomes via a C-terminal domain (EEA1-CT). To identify proteins interacting with EEA1-CT, we screened a human brain library in the yeast two-hybrid system. Fourteen clones reacted strongly with EEA1-CT. Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b. Further two-hybrid analysis suggested that Rab5b also interacts with the N-terminus of EEA1 (EEA1-NT). The interaction of both EEA1-CT and EEA1-NT with Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes. Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b in the two-hybrid system, we detected no interaction with wild-type Rab5a, even though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1. This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar. Instead, we speculate that yeast may contain a GTPase-activating protein (GAP) activity that stimulates Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo.

    European journal of biochemistry 1999;265;1;361-6

  • Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.

    McBride HM, Rybin V, Murphy C, Giner A, Teasdale R and Zerial M

    European Molecular Biology Laboratory, Heidelberg, Germany.

    SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.

    Cell 1999;98;3;377-86

  • Phosphatidylinositol 3-phosphate recognition by the FYVE domain.

    Kutateladze TG, Ogburn KD, Watson WT, de Beer T, Emr SD, Burd CG and Overduin M

    Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262, USA.

    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.

    Molecular cell 1999;3;6;805-11

  • The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer.

    Callaghan J, Simonsen A, Gaullier JM, Toh BH and Stenmark H

    Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein-protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a approximately 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the approximately 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

    The Biochemical journal 1999;338 ( Pt 2);539-43

  • EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

    Simonsen A, Lippé R, Christoforidis S, Gaullier JM, Brech A, Callaghan J, Toh BH, Murphy C, Zerial M and Stenmark H

    Department of Biochemistry, The Norwegian Radium Hospital, Montebello, Oslo.

    GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

    Nature 1998;394;6692;494-8

  • EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

    Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, McCluskey J, Yeo JP, Tock EP and Toh BH

    Department of Pathology, National University of Singapore.

    Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

    The Journal of biological chemistry 1995;270;22;13503-11

Gene lists (3)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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