Membrane insertion of the FYVE domain is modulated by pH - PubMed (original) (raw)

Membrane insertion of the FYVE domain is modulated by pH

Ju He et al. Proteins. 2009 Sep.

Abstract

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 approximately 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.

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Figures

Figure 1

Association of the EEA1 FYVE domain with PtdIns(3)P (PI3P)-containing monolayers and membranes is pH dependent. (a) Insertion of the EEA1 FYVE domain into a POPC/POPE (80:20) monolayer (open symbols) and a POPC/POPE/PtdIns(3)P (77:20:3) monolayer (filled symbols) at pH 6.0 (triangle), 7.4 (circle) and 8.0 (square) monitored as a function of π. (b) The distribution of the EGFP-EEA1 FYVE domain between the cytosol and PtdIns(3)P-enriched vacuolar and endosomal membranes in yeast SEY6210 cells is altered by varying cytosolic pH.

Figure 2

Kinetic and equilibrium SPR analyses of the EEA1 FYVE domain. (a) Equilibrium binding of the EEA1 FYVE domain to the sensor chip coated with POPC/POPE/PtdIns(3)P (78:20:3) was measured at pH 6.0 (▲), 7.4 (●), and 8.0 (■). Binding isotherms were generated from the _R_eq (average of triplicate measurements) versus the protein concentration. Solid line represents theoretical curve constructed from _R_max and _K_d values determined from nonlinear least-squares analysis using the equation _R_eq = _R_max/(1 + K_d/C). (b) Sensorgrams from kinetic experiments show binding of the EEA1 FYVE domain (300 n_M) to POPC/POPE/PtdIns(3)P (77:20:3) vesicles at indicated pH values. The flow rate was maintained at 30 μL/min for both association and dissociation phases. (c) Sensorgrams from kinetic experiments show binding of the wild type, and H1371A and H1372A mutants of the EEA1 FYVE domain to the POPC/POPE/PtdIns(3)P (77:20:3) vesicles at pH 6.0.

Figure 3

The pH-sensitivity is a general feature of the FYVE domain family. (a) The SDS-PAGE gels show the partition of Hrs1, RUFY1, Vps27p, and WDFY1 FYVE domains between the supernatant (s) and PtdIns(3)P-enriched liposome pellet (p) at different pHs. (b) Changes in localization of EGFP-tagged RUFY1, Vps27p, and WDFY1 FYVE domains in HeLa cells upon varying the cytosolic pH. The cells were incubated in solutions buffered to indicated pH prior to visualizing by fluorescence microscopy. (c) The tandem His residues are conserved in the FYVE domain family. Alignment of the FYVE domain sequences: absolutely, moderately, and weakly conserved residues are colored brown, orange, and yellow, respectively. The His residues are in green. Three regions of the FYVE domain involved in coordination of PtdIns(3)P are indicated by black lines above the alignment. (d) The PtdIns(3)P binding pocket defined from the crystal structures of Vps27p FYVE (PDB 1VFY), EEA1 FYVE (PDB 1JOC), and Hrs FYVE (PBD 1DVP). The tandem His residues and Arg/Lys residues located in the R(R/K)HHCRXCG and RVC regions are labeled and colored green and dark gray, respectively.

Figure 4

The tandem His residues are essential for membrane penetration by the FYVE domain. (a) Insertion of the H1371A (filled symbols) and H1372A (open symbols) EEA1 FYVE domain into a POPC/POPE/PtdIns(3)P (78:20:2) monolayer at pH 6.0 (triangle), 7.4 (circle), and 8.0 (square), and into a POPC/POPE (80:20) monolayer at pH 6.0 (diamond). (b) Penetration of the H1371A/H1372A EEA1 FYVE domain into a POPC/POPE/PtdIns(3)P (78:20:2) monolayer at pH 6.0 (triangle), 7.4 (circle), and 8.0 (square) and into a POPC/POPE (80:20) monolayer at pH 6.0 (diamond). (c) pKa values of His1371 and His1372 in the presence and absence of PS-containing DPC micelles were measured by chemical shift perturbation analysis of 1H resonances in NMR spectra. (*) is taken from. (d) Insertion of the p40phox PX domain into POPC/POPE/PtdIns(3)P (77:20:3) and POPC/POPE (80:20) monolayers at pH 6.0, 7.4, and 8.0. (e) Superimposed 1H,15N HSQC spectra of the H1372A EEA1 FYVE domain (0.2 m_M_) collected in the presence of 1.0 m_M_ PtdIns(3)P and 0–50 m_M_ imidazole.

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Membrane insertion of the FYVE domain is modulated by pH - PubMed (original) (raw)