Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch

Abstract

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns3P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns3P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns3P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns3P binding by the FYVE domain in vitro and in vivo. Another PtdIns3P-binding module, the PX domain of Vam7 and p40phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns3P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.

Fig. 1.

pH modulates PtdIns(3)P binding. Superimposed ¹H-¹⁵N HSQC spectra of the PtdIns(3)P-bound (A) and ligand-free (C) FYVE domains collected while pH of the samples was adjusted to values shown (Insets). (B) Superimposed ¹H-¹⁵N HSQC spectra of PtdIns(3)P titration into the FYVE domain sample at a constant pH of 6.8. (D–F) Histograms show normalized (20) ¹⁵N,¹H chemical-shift changes in the FYVE domain backbone amides seen in the corresponding (A–C) spectra. The conserved sequences of the FYVE domain involved in the coordination of PtdIns(3)P are indicated by gray lines in E.(G–I) Residues that exhibit significant resonance perturbations in D–F are labeled and colored in shades of yellow, orange, and red on the FYVE domain surface.

Fig. 2.

pH-dependent membrane targeting by the FYVE domain. The SDS/PAGE gels (A) and histogram (B) show the partition of EEA1 FYVE, Vam7 PX, BSA, and GST between the supernatant (S) and PtdIns(3)P-enriched liposome pellet (P) at different pHs. The experimental points were averaged over three measurements. Changes in localization of the ECFP-FYVE domain (C), EGFP-p40^phox PX domain (D), and EGFP-FYVE-His1371Asn mutant (E) in HeLa cells upon varying the cytosolic pH. Cultures were incubated in solutions buffered to the indicated pH for 15 min. The cells were visualized by fluorescence microscopy. (F) STS-induced apoptosis and acidification of the cytosol. HeLa cells transfected with pECFP-FYVE were incubated for 1 h with or without 1 μM STS.

Fig. 3.

Affinity for PtdIns(3)P increases in acidic media. (A) Superimposed ¹H-¹⁵N HSQC spectra of the FYVE domain recorded during addition of PtdIns(3)P at pH values 6.0, 7.4, and 8.0. The relative concentrations of the ¹⁵N-labeled FYVE domain and PtdIns(3)P (Inset) are color-coded. (B–E) The binding curves used to calculate affinities for PtdIns(3)P based on changes in ¹H and ¹⁵N resonances of the FYVE domain are shown and colored (B) in purple, red, green, orange, blue, brown, and cyan for ¹⁵N resonances (N) of Asn1352, His1372, Ala1349, Asp1351, Arg1369, and ¹H resonances (H) of Ala1349 and Asp1351; (C) in purple, orange, green, brown, and blue for N of His1372, Asp1351, Arg1369, and H of Asp1351 and Arg1369; (D) in purple, green, red, orange, blue, cyan, brown, and magenta for N of Asn1352, Ala1349, His1372, Asp1351, Arg1369, and H of Asp1351, Ala1349, and Arg1369; and (E) in purple, orange, green, red, cyan, blue, brown, and magenta for N of His1371, Asn1352, Ala1349, His1372, and H of His1371, Ala1349, His1372, and Asn1352, respectively. (F) A graph summarizes the dependence of K_d on pH.

Fig. 4.

Protonated His residues are required for the interactions. (A) A model of FYVE domain targeting to PtdIns(3)P-enriched membranes. The FYVE domain binds PtdIns(3)P, inserts its hydrophobic loop (MIL) into the membrane interior, and electrostatically contacts PtdSer. The conserved His and Arg residues of the PtdIns(3)P-binding pocket, as well as MIL and the PtdSer-binding site, are colored in blue, light blue, brown, and green, respectively. PtdIns(3)P and PtdSer are shown as stick models. (B) Schematic diagram showing the PtdIns(3)P head group coordination based on the crystal structure of the EEA1 FYVE domain complexed with 1,3-inositol bisphosphate (6). (C and D) Determining the microscopic ionization constants of His1340, His1371, and His1372 in the absence (C) and in presence (D) of PtdIns(3)P by monitoring perturbations of ¹H resonances in NMR spectra.

Fig. 5.

Molecular mechanism of the EEA1 localization to early endosomes. EEA1 (consists of several indicated domains) is anchored to the endosomal membranes through the specific interaction of its FYVE domain (gray triangle) with PtdIns(3)P (purple), accompanied by hydrophobic insertion (yellow triangle) into the bilayer (light green) and electrostatic interactions with acidic PtdSer (green) headgroups. The protonated state of two adjacent His1371 and His1372 residues (dark blue pentagon with a plus in the center) in the lipid-binding pocket is absolutely required for PtdIns(3)P ligation. A signal is initiated by the phosphorylation of PtdIns at the third position by phosphoinositide 3-kinases (pink). Three membrane proteins, V-type ATPase, Cl^–-channel, and Na⁺/H⁺-exchanger, regulate the acidification of early endosomes.