Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain

Max-Planck-Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
The Journal of Cell Biology (Impact Factor: 9.83). 11/2000; 151(3):601-12.
Source: PubMed

ABSTRACT Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

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Available from: Bernard Hoflack, Sep 25, 2015
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    • "motor protein complexes). Rab5 has been shown to interact with at least 20 identified proteins through the use of Rab5-GST fusion proteins in affinity chromatography preloaded with either GTP or GDP and after binding to effector molecules, eluted with GDP or GTP, respectively[14]: Rabaptin-5[15], RIN3[16], Rabex-5 GEF[17], Rabenosyn-5[18],[19], Rabankyrin-5[20], EEA1[21], PI-3 kinases hVPS34 and p85α-p110β[22],[23], PI-4, PI-5 phosphatases[24], kinesin type motor proteins for microtubule dependent movement[25], APPL1 and APPL2[26], syntaxin-13[27], GGAs[28], Huntingtin and HAP40[29]. The nature of the function of Rab proteins (cycling between the “on” and “off” state) may make for only transient interactions with effectors, which from an experimental perspective, are difficult to trap. "
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    ABSTRACT: Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol (TG) leads to fatty liver disease encompassing steatosis, steatohepatitis and fibrosis. This places individuals at risk of developing cirrhosis, hepatocellular carcinoma or hepatic decompensation and also contributes to the emergence of insulin resistance and dyslipidemias affecting many other organs. Excessive accumulation of TG in adipose tissue contributes to insulin resistance as well as to the release of cytokines attracting leucocytes leading to a pro-inflammatory state. Pathological accumulation of cholesteryl ester (CE) in macrophages in the arterial wall is the progenitor of atherosclerotic plaques and heart disease. Overconsumption of dietary fat, cholesterol and carbohydrates explains why these diseases are on the increase yet offers few clues for how to prevent or treat individuals. Dietary regimes have proven futile and barring surgery, no realistic alternatives are at hand as effective drugs are few and not without side effects. Overweight and obesity-related diseases are no longer restricted to the developed world and as such, constitute a global problem. Development of new drugs and treatment strategies are a priority yet requires as a first step, elucidation of the molecular pathophysiology underlying each associated disease state. The lipid droplet (LD), an up to now overlooked intracellular organelle, appears at the heart of each pathophysiology linking key regulatory and metabolic processes as well as constituting the site of storage of both TGs and CEs. As the molecular machinery and mechanisms of LDs of each cell type are being elucidated, regulatory proteins used to control various cellular processes are emerging. Of these and the subject of this review, small GTPases belonging to the Rab protein family appear as important molecular switches used in the regulation of the intracellular trafficking and storage of lipids.
    05/2014; 28(3):169-77. DOI:10.7555/JBR.28.20140029
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    • "Interestingly, only the combination of both tethers led to significant fusion , suggesting that one cannot substitute for the other. These two tethers also coordinate Rab function with SNARE assembly on early endosomes, because rabenosyn-5 binds the Sec1/Munc18-like Vps45 protein (Nielsen et al. 2000; Morrison et al. 2008), and EEA1 binds "
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    ABSTRACT: Organelles within the endomembrane system are connected via vesicle flux. Along the endocytic pathway, endosomes are among the most versatile organelles. They sort cargo through tubular protrusions for recycling or through intraluminal vesicles for degradation. Sorting involves numerous machineries, which mediate fission of endosomal transport intermediates and fusion with other endosomes or eventually with lysosomes. Here we review the recent advances in our understanding of these processes with a particular focus on the Rab GTPases, tethering factors, and retromer. The cytoskeleton has also been recently recognized as a central player in membrane dynamics of endosomes, and this review covers the regulation of the machineries that govern the formation of branched actin networks through the WASH and Arp2/3 complexes in relation with cargo recycling and endosomal fission.
    Cold Spring Harbor perspectives in biology 03/2014; 6(3). DOI:10.1101/cshperspect.a016832 · 8.68 Impact Factor
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    • "Rab5 is activated by as many as six GEFs including Rabex5 [15], Gapex-5 [16], Rin1 [17], Rin2 [18], [19], Rin3 [20] and Als2 [21] and deactivated by at least two Rab5 GTPase-activating proteins (GAP) RabGap-5 [22] and RN-Tre [23]. Rab5 effectors include EEA1 [24] and Rabinosyn-5 [25], proteins that mediate Rab tethering to membranes through the well-characterized FYVE domain [26], and APPL1 and APPL2 [27], proteins that interact with Rab5 to orchestrate membrane trafficking and that affect gene transcription. PI3 kinase [28] and PI5 and PI4 phosphatases [29] interact with Rab5 to regulate aspects of signal transduction and the temporal regulation of phosphoinositide turnover required for progression of cargo through the early endocytic pathway. "
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    ABSTRACT: Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.
    PLoS ONE 02/2014; 9(2):e90384. DOI:10.1371/journal.pone.0090384 · 3.23 Impact Factor
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