Determinants of Rab5 Interaction with the N Terminus of Early Endosome Antigen 1

Abstract

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...

The Salmonella virulence protein SifA is a G protein antagonist

“…Whether the SKIP PH domain:Rab9 interaction occurs in conjunction with phosphoinositides is not known, and the SKIP PH domain is the first shown to be bound to a RabGTPase. Measured affinities for Rab GTPases and their effectors range from nM to low M K d (21,(26)(27)(28). Further a K d of approximately 10 M has been reported for an analogous interaction between Rac GTPases 1, 2, and 3, and the PH domain of phospholipase C ␤2 protein (PLC-␤2) (29) and more recently a crystal structure of the PLC-␤2 PH:Rac 1 complex has been determined (30).…”

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…Eos Ikaros Pegasus Trps1 (Westman et al, 2004) Gli α helix, Fs 1-2 Intraprotein (Pavletich and Pabo, 1993) Zap1 α helix, Fs 1-2 Intraprotein (Wang et al, 2006) EEA1 β1 of F1 Rab5 (Merithew et al, 2003) Zac1 β1 of F2 Zac1 (Hoffmann et al, 2003) MBP-1 Linker Intraprotein (Omichinski et al, 1992) Forming either self-associations or associations with other members of the family, all members of this family use their C-terminal fingers to mediate PPIs, and use their Nterminal fingers to bind DNA (Hahm et al, 1998;Molnar and Georgopoulos, 1994;Morgan et al, 1997;Perdomo et al, 2000). Trichophinophalageal syndrome 1 (Trps1)…”

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

“…We postulated that the partial colocalization with Rab5 may be due to the phosphatase activity of MTMR2 since many Rab5 effectors require PI(3)P for localizing to Rab5-positive endosomes Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). Therefore we created a MTMR2 phosphatase inactive mutant in the S58A/S631E background and reassessed the ability to colocalize to Rab5-positive and APPL1-positive endosomes.…”

“…Furthermore, Rab5 collaborates with numerous peripheral endosome binding proteins to facilitate fusion of endosome membranes including Rab5 effectors whose endosome targeting requires association with endosomal PI(3)P Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). This property reveals that the endosomal activities of Rab5 can be dramatically affected by PI(3)P levels.…”

“…This property reveals that the endosomal activities of Rab5 can be dramatically affected by PI(3)P levels. For example, the critical endosomal effectors Rabenosyn-5 and Early Endosomal Antigen 1 (EEA1) are essential tethering factors important for endosome fusion events (Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). These effectors require association with Rab5 and PI(3)P simultaneously to localize to early endosomes.…”