Multivalent Endosome Targeting by Homodimeric EEA1

Dumas, John J.; Merithew, Eric Lee; Eathiraj, Sudharshan; Rajamani, Deepa; Hayes, Stephen F; Lawe, Deirdre C.; Corvera, Silvia; Lambright, David G.

doi:10.1016/s1097-2765(01)00385-9

Cited by 234 publications

(295 citation statements)

References 36 publications

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“…This would not affect the level of membrane-bound EEA1. EEA1 was reported to form a parallel dimer in cells [28,29], which was proposed to be the functional unit of an EEA1 tethering complex. Thus, more EEA1 dimerization could explain the increased endosome clustering and enlargement phenotype associated with the loss of p97 function.…”

Section: Inhibition Of P97 Causes Increased Eea1 Oligomerizationmentioning

confidence: 99%

“…The carboxyl-terminus of EEA1 carries a FYVE domain responsible for binding to phosphatidyl inositol-3-phosphate on the membrane [19,20,[26][27][28]. The N-terminal C2H2 zinc finger domain and a region upstream of the FYVE domain serve as Rab5-binding sites [21].…”

Section: Introductionmentioning

confidence: 99%

“…EEA1 also harbors a calmodulin-binding (IQ) domain and a long helical segment [17]. The latter is responsible for the homodimerization of EEA1, which is thought to be a prerequisite for the tethering function [28,29]. In cells, Rab5 activation leads to the complex of the EEA1 dimer into a large protein complex that contains other Rab5 effectors such as the Rab5 GTP exchange factor Rabex-5 and Rabaptin-5 as well as the endosome-specific t-SNARE Syntaxin-13, which results in the fusion of endosomes [24].…”

Section: Introductionmentioning

confidence: 99%

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The p97 ATPase associates with EEA1 to regulate the size of early endosomes

Ramanathan

2011

Cell Res

100

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The AAA (ATPase-associated with various cellular activities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD). In membrane fusion, p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimidesensitive fusion protein), which promotes membrane fusion by disassembling a SNARE complex. In ERAD, p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome. Here, we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate. We demonstrate that a fraction of p97 is localized to the early endosome membrane, where it binds EEA1 via the N-terminal C2H2 zinc finger domain. Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes, which is associated with an altered trafficking pattern for an endocytic cargo. Mechanistically, we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane. We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

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Section: Inhibition Of P97 Causes Increased Eea1 Oligomerizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The p97 ATPase associates with EEA1 to regulate the size of early endosomes

Ramanathan

2011

Cell Res

100

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“…Together, the FYVE domains form a flat surface orthogonal to the coiled coil domain. Residues that contact the lipid head group are on the face of the protein opposite the coiled coil, and four hydrophobic residues near the phosphate binding residues form a loop that would dip down into the hydrophobic core of the bilayer (88). Thus, in EEA1, specificity of the head groupbinding pocket is supplemented by nonspecific hydrophobic interactions as well as strengthened by being bivalent.…”

Section: Fyve Domainsmentioning

confidence: 99%

Phosphoinositides in Constitutive Membrane Traffic

Roth

2004

Physiological Reviews

265

267

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Proteins that make, consume, and bind to phosphoinositides are important for constitutive membrane traffic. Different phosphoinositides are concentrated in different parts of the central vacuolar pathway, with phosphatidylinositol 4-phosphate predominate on Golgi, phosphatidylinositol 4,5-bisphosphate predominate at the plasma membrane, phosphatidylinositol 3-phosphate the major phosphoinositide on early endosomes, and phosphatidylinositol 3,5-bisphosphate found on late endocytic organelles. This spatial segregation may be the mechanism by which the direction of membrane traffic is controlled. Phosphoinositides increase the affinity of membranes for peripheral membrane proteins that function for sorting protein cargo or for the docking and fusion of transport vesicles. This implies that constitutive membrane traffic may be regulated by the mechanisms that control the activity of the enzymes that produce and consume phosphoinositides. Although the lipid kinases and phosphatases that function in constitutive membrane traffic are beginning to be identified, their regulation is poorly understood.

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“…This zinc-stabilized module is found in 29 human proteins and has been engineered into an intracellular probe having nanomolar PtdIns(3)P affinity (21,22). Three-dimensional structures of the FYVE domains of EEA1 (13,23), Hrs (24), and Vps27 (25) proteins have been solved. Despite the structural similarity, different models of their membrane orientations have been inferred, emphasizing the need for experimentally derived estimates of membrane insertion.…”

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confidence: 99%

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Kutateladze

Capelluto

Ferguson

et al. 2004

Journal of Biological Chemistry

109

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Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.Cellular processes including signal transduction, vesicular trafficking, and cytoskeletal rearrangement require selective recruitment of proteins to membrane surfaces. Well established mechanisms for localizing cytosolic proteins to membranes include electrostatic interactions through a basic peptide sequence, anchoring by covalently attached acyl chains, and association with the cytoplasmic domains of transmembrane proteins (reviewed in Refs. 1 and 2). (4), PDZ (5), and PTB (6) domains. Although the majority of these domains can interact with several PIs, the FYVE domain is remarkably selective for PtdIns(3)P (7-9). In addition to PI ligation, these domains often insert hydrophobic elements into the membrane bilayer, as has been demonstrated for the C2 (10), ENTH (11), FERM (12), FYVE (13), and PX (14) domains and the vinculin tail (15). Insertion into the membrane can be accompanied by interactions with multiple lipid head groups. For example, the PX domain of the p47 subunit of the NADPH oxidase binds cooperatively to PtdIns(3)P and phosphatidic acid (16), and the vinculin tail co-ligates phosphatidylinositol 4,5-bisphosphate and PtdSer (15). Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17-20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipi...

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Multivalent Endosome Targeting by Homodimeric EEA1

Cited by 234 publications

References 36 publications

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

Phosphoinositides in Constitutive Membrane Traffic

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

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