Hrs recruits clathrin to early endosomes

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.     

Early endosomal localization of hrs requires a sequence within the proline- and glutamine-rich region but not the FYVE finger

Hrs is an early endosomal protein that is tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in endocytosis of growth factor-receptor complexes through early endosomes. Early endosomal localization of Hrs seems to be essential for Hrs to exert its function in the endocytosis. Hrs has a FYVE finger domain that binds specifically to phosphatidylinositol 3-phosphate in vitro. The FYVE finger is a likely domain that mediates membrane association of endosomal proteins. In this study, we examined whether the FYVE finger participates in early endosomal targeting of Hrs. Hrs with a zinc binding-defective FYVE finger was still localized to early endosomes. In addition, the N-terminal FYVE finger-containing fragment of Hrs showed a cytosolic distribution in mammalian cells. These results indicate that the FYVE finger is not required for the localization of Hrs to early endosomes. Furthermore, by analyzing a series of deletion mutants of Hrs, we identified a sequence of about 100 amino acids within the C-terminal proline- and glutamine-rich region as a domain essential for the targeting of Hrs to early endosomes.     

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P‐enriched membranes and membrane‐mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ～24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P‐containing membranes in vitro and in vivo, indicating that pH‐dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH‐sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid‐bound form, promoting its penetration and increasing the membrane residence time. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Endofin recruits TOM1 to endosomes

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.     

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

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

Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P(2), thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol ( approximately 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) ( approximately 20%), and plasma membrane (( approximately )4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.     

Time-resolved Ultrastructural Detection of Phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization. The probe localized to early and late endocytic compartments according to the time period of dimerization, which indicates that PtdIns(3)P is turned over via the endocytic machinery. In the functional context of epidermal growth factor (EGF) stimulation, we observed that dimerization of the probe led to clustering of mainly early endocytic structures, leaving most of the probe localized to the limiting membrane of endosomes. Interestingly, these clustered endosomes contained coats positive for the PtdIns(3)P-binding protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), indicating that the probe did not displace Hrs binding. We conclude that the dimerizer-inducible probe is useful for the time-resolved detection of PtdIns(3)P at the ultrastructural level, but its effects on endosome morphology after EGF stimulation need to be taken into account. (J Histochem Cytochem 58:1025–1032, 2010)     

The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.     

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.     

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

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.     

Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.     

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.     

Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.     

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

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

Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization.

PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.     

A WD-FYVE protein binds to the kinases Akt and PKCzeta/lambda

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.     

Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.     

Vps27 recruits ESCRT machinery to endosomes during MVB sorting

Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.     

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.     

Phosphatidylinositol 3-phosphate recognition by the FYVE domain.

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