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.

The Rab5 GTPase, an essential regulator of endocytosis and endosome biogenesis, is activated by guanine-nucleotide exchange factors (GEFs) that contain a Vps9 domain. Here, we show that the catalytic core of the Rab GEF Rabex-5 has a tandem architecture consisting of a Vps9 domain stabilized by an indispensable helical bundle. A family-wide analysis of Rab specificity demonstrates high selectivity for Rab5 subfamily GTPases. Conserved exchange determinants map to a common surface of the Vps9 domain, which recognizes invariant aromatic residues in the switch regions of Rab GTPases and selects for the Rab5 subfamily by requiring a small nonacidic residue preceding a critical phenylalanine in the switch I region. These and other observations reveal unexpected similarity with the Arf exchange site in the Sec7 domain.

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.Protein trafficking in the secretory and endocytic pathways requires the coordinated participation of distinct protein classes, among which are tethering proteins and Rab GTPases. In the endocytic pathway the small GTPase Rab5 has been proposed to play key roles in clathrin-coated vesicle formation, the regulation of endosome motility, and early endosome fusion (1, 2). The specific mechanisms by which these steps are regulated by Rab5 is not fully understood, but the regulation of endosome fusion by Rab5 appears to be dependent on the actions of the cytoplasmic protein EEA1.1 EEA1 in turn has been postulated to mediate the obligatory docking or tethering steps required prior to homotypic endosome fusion, as well as to participate in the fusion process itself (3, 4).EEA1 is a large ϳ170-kDa polypeptide that contains extensive regions of coiled-coil as well as a conserved Zn 2ϩ finger motif at its extreme COOH terminus, called the FYVE domain (5). The FYVE domain interacts specifically with PtdIns-3-P, the most abundant product of PI3-kinase activity in eukaryotic cells (6, 7), and is crucial for the interaction of EEA1 with endosomal membranes (8). The dual interaction of EEA1 with PtdIns-3-P and Rab5 has been hypothesized to confer increased specificity and stability to the association of EEA1 with endosomal membranes (3, 4). To directly test this hypothesis we have identified critical residues required for the interaction of the EEA1 COOH-termin...

Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central ␤ sheet and the ␣2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.As general regulators of intracellular vesicle transport between donor and acceptor membranes, Rab proteins comprise the largest GTPase family with 11 distinct homologues in yeast and more than 50 known Rab GTPases in mammals (3-7). As is true of other GTPases of the Ras superfamily, Rabs cycle between active (GTP-bound) and inactive (GDP-bound) conformations (3,8,9). A key question concerns the molecular and structural mechanisms by which Rab GTPases generate specificity for a diverse spectrum of effectors and regulatory factors. Biochemical and genetic studies of chimeric and mutant Rab proteins have identified several hypervariable regions, including the N and C termini and the ␣3/␤5 loop, that play an important role in determining functional specificity (10, 11). However, interactions involving hypervariable regions cannot explain the ability of Rab GDI 1 (GDP dissociation inhibitor) to recognize most or all Rab GTPases yet still discriminate against other GTPase families or the ability of certain regulatory factors to recognize particular Rab subfamilies (12-15). These observations imply the existence of specificity determinants that are common to all Rab GTPases but not other GTPase families, as well as determinants that are conserved only within particular Rab subfamilies.Crystallographic studies of Rab GTPases have identified structural motifs a...

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.As master regulators of membrane trafficking, Rab GTPases cycle between active (GTP-bound) and inactive (GDP-bound) conformations (1-3). In the active conformation, Rab GTPases interact with diverse effectors implicated in vesicle budding, cargo sorting, motor-dependent transport, tethering, docking, and fusion. Guanine nucleotide exchange factors, GTPase-activating proteins, and other accessory factors, including Rab GDP dissociation inhibitor, provide multiple points of regulation throughout the GTPase cycle by modulating nucleotide binding, GTP hydrolysis, and membrane association (4). Protein kinases and phosphatases have also been implicated in the regulation of Rab function, either directly or by phosphorylation of effectors and regulatory factors (5-8). Thus, through regulated interactions with effectors, Rab GTPases couple signal transduction networks to the membrane trafficking machinery.Both fluid phase and receptor-mediated endocytosis depend on activation of Rab5, which plays a critical role in clathrincoated vesicle formation, endosome motility, and early endosome fusion (9). Activated Rab5 interacts with diverse effectors, including scaffolding proteins and tethering factors, and further influences signaling and trafficking events by recruitment of class I and III phosphoinositide 3-kinases to endosomes (10 -14). The class III phosphoinositide 3-kinase, hVPS34, selectively generates phosphatidylinositol 3-phosphate, PtdIns...