The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Marino Zerial - One of the best experts on this subject based on the ideXlab platform.

  • regulation of egfr signal transduction by analogue to digital conversion in Endosomes
    eLife, 2015
    Co-Authors: Roberto Villasenor, Hidenori Nonaka, Yannis Kalaidzidis, Perla Del Contezerial, Marino Zerial
    Abstract:

    An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in Endosomes. Cells respond to higher EGF concentrations by increasing the number of Endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between Endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in Endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

  • mammalian corvet is required for fusion and conversion of distinct early endosome subpopulations
    Traffic, 2014
    Co-Authors: Enrico D Perini, Ramona Schaefer, Martin Stoter, Yannis Kalaidzidis, Marino Zerial
    Abstract:

    Early Endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late Endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early Endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 Endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive Endosomes but not EEA1 Endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 Endosomes indicative of another role in the conversion of EEA1 Endosomes into late Endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.

  • rab conversion as a mechanism of progression from early to late Endosomes
    Cell, 2005
    Co-Authors: Jochen C Rink, Yannis Kalaidzidis, Eric Ghigo, Marino Zerial
    Abstract:

    Summary The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab5 and Rab7 are key determinants of early and late Endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab5 dynamically fluctuates on individual early Endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger Endosomes that migrate from the cell periphery to the center where Rab5 is rapidly replaced with Rab7. The class C VPS/HOPS complex, an established GEF for Rab7, interacts with Rab5 and is required for Rab5-to-Rab7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late Endosomes.

  • rhod regulates endosome dynamics through diaphanous related formin and src tyrosine kinase
    Nature Cell Biology, 2003
    Co-Authors: Stephane Gasman, Yannis Kalaidzidis, Marino Zerial
    Abstract:

    Early Endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early Endosomes. Expression of RhoD and hDia2C induces a striking alignment of early Endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early Endosomes through interactions with the actin cytoskeleton.

  • distinct membrane domains on Endosomes in the recycling pathway visualized by multicolor imaging of rab4 rab5 and rab11
    Journal of Cell Biology, 2000
    Co-Authors: Birte Sonnichsen, Erik Nielsen, Stefano De Renzis, Jens Rietdorf, Marino Zerial
    Abstract:

    Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on Endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that Endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

Jean Gruenberg - One of the best experts on this subject based on the ideXlab platform.

  • ion flux and the function of Endosomes and lysosomes ph is just the start the flux of ions across endosomal membranes influences endosome function not only through regulation of the luminal ph
    BioEssays, 2011
    Co-Authors: Cameron C. Scott, Jean Gruenberg
    Abstract:

    The ionic nature of Endosomes varies considerably in character along the endocytic pathway. Counter-ion flux across the limiting membrane of Endosomes has long been considered essential for full acidification and normal endosome/lysosomal function. The proximal functions of luminal ions, however, have been difficult to assess. The recent development of transgenic mice carrying mutations in the intracellular chloride channels (ClCs) has provided a tool to uncouple Cl(-) influx from endosomal acidification. Intriguingly, many of the defects of the endo-lysomal system attributed to aberrant pH persist in the Cl(-)-deficient mice implying a direct regulatory role for Cl(-) influx in endosome function. These observations may begin to explain the abundance of endosomal ion transporters, including ClCs, sodium-proton exchangers, two-pore channels and mucolipins, that have been localized to endo-lysosomes, and the extensive changes in luminal ion composition therein. In this review, we summarize what is known regarding the mediators of endosomal ion flux, and discuss the implications of changing ionic content on endo-lysosomal function.

  • Annexin A2-dependent polymerization of actin mediates endosome biogenesis
    Developmental cell, 2009
    Co-Authors: Etienne Morel, Robert G. Parton, Jean Gruenberg
    Abstract:

    Early Endosomes give rise to multivesicular intermediates during transport toward late Endosomes. Much progress has been made in understanding the sorting of receptors into these intermediates, but the mechanisms responsible for their biogenesis remain unclear. Here, we report that F-actin is necessary for transport beyond early Endosomes and endosome formation. We found that Endosomes captured by actin cables were essentially stationary, but early Endosomes also exhibited patches of F-actin and facilitated selective F-actin nucleation and polymerization. Our data show that nucleation of actin patches by early Endosomes is strictly dependent on annexin A2, a protein involved in early-to-late endosome transport. It also requires the actin nucleation factor Spire1 and involves Arp2/3, which is needed for filament branching. We conclude that actin patches are nucleated on early Endosomes via annexin A2 and Spire1, and that these patches control endosome biogenesis, presumably by driving the membrane remodeling process.

  • the escrt i subunit tsg101 controls endosome to cytosol release of viral rna
    Traffic, 2008
    Co-Authors: Pierrephilippe Luyet, Thomas Falguieres, Veronique Pons, Asit K Pattnaik, Jean Gruenberg
    Abstract:

    Like other enveloped viruses, vesicular stomatitis virus infects cells through Endosomes. There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Previously, we reported that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular Endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late Endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. In this study, we show that the tumor susceptibility gene 101 (Tsg101) subunit of the endosomal sorting complexes required for transport (ESCRT)-I complex, which mediates receptor sorting into multivesicular Endosomes, is dispensable for viral envelope fusion with endosomal membranes and viral RNA transport to late Endosomes but is necessary for infection. Our data indicate that Tsg101, in contrast to the ESCRT-0 component Hrs, plays a direct role in nucleocapsid release from within multivesicular Endosomes to the cytoplasm, presumably by controlling the back-fusion process. We conclude that Tsg101, through selective interactions with its partners including Hrs and Alix, may link receptor sorting and lysosome targeting to the back-fusion process involved in viral capsid release.

  • in vitro budding of intralumenal vesicles into late Endosomes is regulated by alix and tsg101
    Molecular Biology of the Cell, 2008
    Co-Authors: Thomas Falguieres, Cameron C. Scott, Pierrephilippe Luyet, Christin Bissig, Marieclaire Velluz, Jean Gruenberg
    Abstract:

    Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late Endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.

  • the recycling endosome of madin darby canine kidney cells is a mildly acidic compartment rich in raft components
    Molecular Biology of the Cell, 2000
    Co-Authors: Raluca Gagescu, Robert G. Parton, Nicolas Demaurex, Walter Hunziker, Lukas A Huber, Jean Gruenberg
    Abstract:

    We present a biochemical and morphological characterization of recycling Endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling Endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling Endosomes are distinct from conventional early Endosomes. We also find that recycling Endosomes are less acidic than early Endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling Endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling Endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling Endosomes and early Endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Silvia Corvera - One of the best experts on this subject based on the ideXlab platform.

  • rabenosyn 5 defines the fate of the transferrin receptor following clathrin mediated endocytosis
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Deanna M Navaroli, Kevin E. Fogarty, David G. Lambright, Lawrence M. Lifshitz, Karl D Bellve, Clive Standley, James Cardia, Deborah M Leonard, Silvia Corvera
    Abstract:

    Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early Endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early Endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of Endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of Endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

  • tgfβ receptor internalization into eea1 enriched early Endosomes role in signaling to smad2
    Journal of Cell Biology, 2002
    Co-Authors: Susan J Hayes, Anil Chawla, Silvia Corvera
    Abstract:

    Transforming growth factor (TGF)beta is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGF beta receptors internalize into Endosomes containing the early endosomal protein EEA1. The extent of TGF beta-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGF beta-stimulated transcription correlated closely with the extent of internalization of the receptor. TGF beta signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn(++) finger motif. Here we show that SARA localizes to Endosomes containing EEA1, and that disruption of this localization inhibits TGF beta-induced Smad2 nuclear translocation. These results indicate that traffic of the TGF beta receptor into the endosome enables TGF beta signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.

  • Sequential Roles for Phosphatidylinositol 3-Phosphate and Rab5 in Tethering and Fusion of Early Endosomes via Their Interaction with EEA1
    The Journal of biological chemistry, 2001
    Co-Authors: Deirdre C Lawe, Kevin E. Fogarty, David G. Lambright, Richard A. Tuft, John J. Dumas, Eric Lee Merithew, Anil Chawla, Walter A. Carrington, Lawrence M. Lifshitz, Silvia Corvera
    Abstract:

    Abstract 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.

Gia K Voeltz - One of the best experts on this subject based on the ideXlab platform.

  • a novel class of er membrane proteins regulates er associated endosome fission
    Cell, 2018
    Co-Authors: Melissa J Hoyer, Patrick J Chitwood, Gia K Voeltz, Christopher C Ebmeier, Jonathan F Striepen, William M Old
    Abstract:

    Endoplasmic reticulum (ER) membrane contact sites (MCSs) mark positions where Endosomes undergo fission for cargo sorting. To define the role of ER at this unique MCS, we targeted a promiscuous biotin ligase to cargo-sorting domains on endosome buds. This strategy identified the ER membrane protein TMCC1, a member of a conserved protein family. TMCC1 concentrates at the ER-endosome MCSs that are spatially and temporally linked to endosome fission. When TMCC1 is depleted, endosome morphology is normal, buds still form, but ER-associated bud fission and subsequent cargo sorting to the Golgi are impaired. We find that the endosome-localized actin regulator Coronin 1C is required for ER-associated fission of actin-dependent cargo-sorting domains. Coronin 1C is recruited to endosome buds independently of TMCC1, while TMCC1/ER recruitment requires Coronin 1C. This link between TMCC1 and Coronin 1C suggests that the timing of TMCC1-dependent ER recruitment is tightly regulated to occur after cargo has been properly sequestered into the bud.

  • er contact sites define the position and timing of endosome fission
    Cell, 2014
    Co-Authors: Ashley A Rowland, Patrick J Chitwood, Melissa J Phillips, Gia K Voeltz
    Abstract:

    Summary Endocytic cargo and Rab GTPases are segregated to distinct domains of an endosome. These domains maintain their identity until they undergo fission to traffic cargo. It is not fully understood how segregation of cargo or Rab proteins is maintained along the continuous endosomal membrane or what machinery is required for fission. Endosomes form contact sites with the endoplasmic reticulum (ER) that are maintained during trafficking. Here, we show that stable contacts form between the ER and endosome at constricted sorting domains, and free diffusion of cargo is limited at these positions. We demonstrate that the site of constriction and fission for early and late Endosomes is spatially and temporally linked to contact sites with the ER. Lastly, we show that altering ER structure and dynamics reduces the efficiency of endosome fission. Together, these data reveal a surprising role for ER contact in defining the timing and position of endosome fission. PaperFlick

  • endoplasmic reticulum endosome contact increases as Endosomes traffic and mature
    Molecular Biology of the Cell, 2013
    Co-Authors: Jonathan R. Friedman, Ashley A Rowland, Jared R. Dibenedetto, Matthew West, Gia K Voeltz
    Abstract:

    The endosomal pathway is responsible for plasma membrane cargo uptake, sorting, and, in many cases, lysosome targeting. Endosome maturation is complex, requiring proper spatiotemporal recruitment of factors that regulate the size, maturity, and positioning of endosomal compartments. In animal cells, it also requires trafficking of Endosomes on microtubules. Recent work has revealed the presence of contact sites between some Endosomes and the endoplasmic reticulum (ER). Although these contact sites are believed to have multiple functions, the frequency, dynamics, and physical attributes of these contacts are poorly understood. Here we use high-resolution three-dimensional electron microscopy to reveal that ER tubules wrap around Endosomes and find that both organelles contact microtubules at or near membrane contact sites. As Endosomes traffic, they remain bound to the ER, which causes the tubular ER to rearrange its structure around dynamic Endosomes at contact sites. Finally, as Endosomes transition through steps of maturation, they become more tightly associated with the ER. The major implication of these results is that Endosomes mature and traffic while coupled to the ER membrane rather than in isolation.

Harald Alfred Stenmark - One of the best experts on this subject based on the ideXlab platform.

  • Protein sorting into multivesicular Endosomes.
    Current opinion in cell biology, 2003
    Co-Authors: Camilla Raiborg, Tor Erik Rusten, Harald Alfred Stenmark
    Abstract:

    Multivesicular Endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular Endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular Endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular Endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular Endosomes to the plasma membrane to facilitate viral budding.

  • localization of phosphatidylinositol 3 phosphate in yeast and mammalian cells
    The EMBO Journal, 2000
    Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred Stenmark
    Abstract:

    Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to Endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early Endosomes and in the internal vesicles of multivesicular Endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of Endosomes and vacuoles of wild-type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

  • localization of phosphatidylinositol 3 phosphate in yeast and mammalian cells
    The EMBO Journal, 2000
    Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred Stenmark
    Abstract:

    Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to Endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early Endosomes and in the internal vesicles of multivesicular Endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of Endosomes and vacuoles of wild-type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

  • Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.
    The EMBO journal, 2000
    Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred Stenmark
    Abstract:

    Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to Endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early Endosomes and in the internal vesicles of multivesicular Endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of Endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

  • rabaptin 5 is a direct effector of the small gtpase rab5 in endocytic membrane fusion
    Cell, 1995
    Co-Authors: Harald Alfred Stenmark, Gaetano Vitale, Oliver Ullrich, Marino Zerial
    Abstract:

    Abstract We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early Endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged Endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early Endosomes. Rab5 recruits rabaptin-5 to purified early Endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.