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

Lee et al. 10.1073/pnas.0503900102.

Supporting Information

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Supporting Text
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11





Fig. 6. Superimposed 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the PtdIns(3)P-bound FYVE domain color-coded according to the pH values indicated (Inset).





Fig. 7. Titration curves illustrate dependence of the 1H and 15N chemical shifts of the PtdIns(3)P-bound (A) and the ligand-free (B) FYVE domain on pH. Small arrows indicate inflection points estimated by nonlinear fitting using a modified Henderson-Hasselbalch equation.





Fig. 8. Superimposed 1H-15N HSQC spectra of the PX domain bound to PtdIns(3)P recorded at the pH values depicted (Inset).





Fig. 9. Superimposed 1H-15N HSQC spectra of the His1371Tyr (A) and His1372Lys (B) mutant FYVE domains show no resonance perturbations during C4-PtdIns(3)P titration, indicating that these His residues are required for the lipid binding.





Fig. 10. Mutation of the His1371 or His1372 residues disrupts in vivo localization of EGFP-EEA1 FYVE to endosomes. Each of the His switch residues were changed to Asn by site-directed mutagenesis and expressed in wild-type yeast cells as fusions to EGFP. The dotted lines outline each of the cells.





Fig. 11. The FYVE domain insertion into dodecyl phosphocholine (DPC) micelles and electrostatic interaction with PtdSer are not pH-dependent. (A) A model of sequential interactions of the FYVE domain with C4-PtdIns(3)P or DPC micelles involves the indicated monovalent affinities at pH 6.0 (top numbers) and pH 6.8 (bottom numbers in square brackets). Titration of C4-PtdIns(3)P (B and C) and a mixture of d38-DPC and 10%PtdSer (D and E) into the FYVE domain induces changes in the backbone 15N and 1H resonances, as observed in 1H-15N HSQC spectra. The 15N-labeled FYVE domain was initially either unbound (B and D), micelle-associated (C), or C4-PtdIns(3)P-bound (E). The binding curves are 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 Ala1351; (C) in blue, orange, red, green, and magenta for N of Gln1375, Val1368, Ala1389, and H of Val1368 and Ala1389; (D) in green, red, blue, cyan, magenta, and brown for N of Thr1367, Val1366, Arg1369, Val1354, and H of Val1354 and Arg1369; (E) in purple, orange, cyan, brown, red, green, blue, and violet for N of Thr1367, Val1366, Val1368, Ser1365, Phe1364, and H of Val1366, Val1368, and Ser1365, respectively. The estimated binding affinities are inset and shown in A.





Supporting Text

Mutagenesis. Single and double mutations of the EEA1 FYVE were generated by using QuikChange (Stratagene). The following mutants were generated: H1371A, H1371K, H1371N, H1371R, H1371Y, H1372A, H1372K, H1372N, H1372R, H1372Y, H1371A/H1372A, H1371Y/H1372Y, and H1371K/H1372K. The sequences of all mutant proteins were confirmed by DNA sequencing.

Protein Expression and Purification. DNA fragments encoding residues 1325-1410 of human EEA1 FYVE were cloned in a pGEX-KG vector (Amersham Pharmacia). The PX domain construct comprising residues 2-122 of Vam7 was produced as described in ref. 1. The FYVE and PX domains were expressed in the Escherichia coli BL21 (DE3) pLysS or BL21 Codon Plus RP strains in M9 media supplemented with 15NH4Cl or in LB and purified on glutathione Sepharose 4B beads (Amersham Pharmacia) as described (1, 2). The proteins were concentrated into 20 mM d11-Tris, pH 6.8, in the presence of 100 or 200 mM KCl, 1 or 20 mM perdeuterated DTT, 0 or 50 mM 4-amidinophenylmethane sulfonyl fluoride, 1 mM NaN3, and 7 or 99.996% 2H2O.

Calculations of pKa values. The pKa values of His1340, His1371, and His1372 of the EEA1 FYVE domain were measured based on the pH-dependent chemical shift changes of the histidines’ Hd2 and He1 resonances. The 1D 1H NMR spectra of 0.5 mM unlabeled FYVE domain were recorded in the presence and absence of 2.5 mM C4-PtdIns(3)P in 2H2O while pH of the samples was adjusted stepwise from 4.7 to 9.4. Measurements of the sample pH were not corrected for deuterium isotope effects. The histidine resonances were assigned based on fully assigned spectra of the FYVE domain recorded at pH 6.8 (ref. (13)). Proton chemical shifts were calibrated and referenced to the internal standard, 2,2-dimethyl-2-silapentane-5-sulfonate (Sigma).

The microscopic ionization constants of His residues and the phenomenological ionization constant describing the dependence of the PtdIns(3)P-FYVE domain interaction upon pH were obtained by a nonlinear least squares fitting of the chemical shifts observed in 1D and 2D spectra as a function of pH. The Henderson-Hasselbalch equation: dobs = [dacid + dbase10(pH-pKa)]/[1 + 10(pH-pKa)] was modified for the fast exchange processes on the NMR time scale. The dobs value is chemical shift at a given pH, and dacid and dbase are chemical shifts at the low and high extremes of pH, respectively.

Estimation of the lipid binding affinities. Lipid binding was characterized by monitoring chemical shift changes in 1H-15N HSQC spectra of 0.2 mM FYVE domain as C4- phosphatidylinositol 3-phosphate [PtdIns(3)P] (up to 1.6 mM) or a mixture of d38-dodecylphosphocholine (DPC) (Cambridge Isotope Laboratories, Cambridge, MA) with 10% wt/wt 1,2-dicaproyl-PtdSer (Avanti Polar Lipids) (up to 250 mM) were added stepwise to the NMR sample. Micellar concentration corresponds to the solution concentration of intact micelles and is obtained by dividing the value of a detergent molecular concentration by an average aggregation number. Kd values were estimated by a nonlinear least-squares analysis using the xmgr program and the equation Dd = Ddmax((([L]+[P]+Kd)-sqrt(([L]+[P]+Kd)2+(4*[P]*[L])))/(2*[P])), where [L] is concentration of the lipid, [P] is concentration of the protein, Dd is the observed chemical shift change, and Ddmax is the chemical-shift change at saturation.

The in vivo localization of the EGFP-fusion FYVE proteins in yeast cells. The EGFP-EEA1-FYVE domain yeast expression plasmid was generated as described (3). Site-directed mutagenesis was used to change the codons encoding His1371 and His1372 to Asn by using a QuikChange kit (Stratagene). The expression plasmids were transformed into a wild-type yeast strain (SEY6210: MATa, his3-D200, ura3-52, trp1-D901, lys2-801, suc2-D9, and leu2-3,112). For microscopy, yeast strains were grown in selective media overnight and the next morning, a fresh culture was inoculated and grown at 26°C until an OD600 of 0.5. The cells were visualized by fluorescence microscopy as described (2) using a Nikon Eclipse E800 microscope fitted with a cooled high-resolution charge-coupled device camera (Hamamatsu Photogenics, Hamamatsu City, Japan). Images were acquired by using phase 3 imaging software (Phase 3 Imaging Systems, Glen Mills, PA).

Liposome binding. The liposome-binding assays were performed according to a method modified from ref. 4. Briefly, solutions of PtdCho, PtdEtn, PtdSer (Avanti Polar Lipids), PtdIns, and PtdIns(3)P (Echelon, Salt Lake City, UT) dissolved in CHCl3:MeOH:H2O (65:25:4) were mixed and dried down under vacuum. The lipids were resuspended in 20 mM Tris, 100 mM KCl (pH 7.0) and sonicated for 15 min in a bath sonicator. Liposomes were collected by centrifugation at 25,000 × g for 10 min and resuspended to a final concentration of 4 mM total lipids in 100 ml of 20 mM Tris/100 mM KCl buffer. The pH of the suspensions was adjusted to the indicated values. Liposomes were incubated with 10 mg of GST-EEA1 FYVE, Vam7 PX, GST, or BSA (Fisher) for 15 min at room temperature and then collected again by centrifugation. The liposome pellets were resuspended in 100 ml of buffer and analyzed using SDS/PAGE with Coomassie brilliant blue staining. The amount of protein on the gel was estimated by densitometric analysis using scion image software (Scion. Frederick, MD).

  1. Cheever, M. L., Sato, T. K., de Beer, T., Kutateladze, T. G., Emr, S. D. & Overduin, M. (2001) Nat. Cell. Biol.3, 613-618.
  2. Kutateladze, T. G., Ogburn, K. D., Watson, W. T., de Beer, T., Emr, S. D., Burd, C. G. & Overduin, M. (1999) Mol. Cell3, 805-811.
  3. 3. Burd, C. G. & Emr, S. D. (1998) Mol. Cell2, 157-162.

This Article

  1. Published online before print September 2, 2005, doi: 10.1073/pnas.0503900102 PNAS September 13, 2005 vol. 102 no. 37 13052-13057
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