Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity

Abstract

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.

Fig. 1. FtsY and FtsY–NG bind to liposomes composed of E.coli phospholipids. Purified FtsY or FtsY–NG (250 ng) was incubated for 10 min at 37°C in the absence (lanes 1–4) or presence (lanes 5–8) of 50 μg of liposomes prepared from E.coli lipids in 50 mM HEPES pH 7.6, 0.5 M KOAc, 5 mM Mg(OAc)₂ in a final volume of 25 μl. Samples were subjected to floatation gradient centrifugation as described in Materials and methods. The gradient was collected in four fractions from the top. Fractions were TCA precipitated and analysed by immunoblotting using affinity-purified αFtsY serum. The lower band of the FtsY doublet represents a degradation product that lacks the 14 N–terminal amino acids (Luirink et al., 1994).

Fig. 2. FtsY–induced aggregation of lipid vesicles suggests two binding sites. (A) Absorbance changes (at 405 nm) induced by the addition of 0.2 μM FtsY to lipid vesicles composed of PE:PG at the indicated molar fractions. The lipid:protein molar ratio was 500. (B) The same as (A), but with 0.2 μM FtsY–NG. (C) Absorbance increase versus molar fraction of the anionic lipid (PG) for three different concentrations of FtsY (open circles, 0.05 μM; dotted circles, 0.1 μM; filled circles, 0.2 μM). The lipid concentration was always 100 μM. Inset: dose dependence of FtsY–induced aggregation of PG:PE 1:1 LUV.

Fig. 3. FtsY (A) and FtsY–NG (B) insert into synthetic lipid monolayers. The increase in surface pressure upon injection of the protein into the subphase was measured as a function of the initial surface pressure. Monolayers of DOPG, DOPE and DOPC were used.

Fig. 4. Insertion of FtsY (A) and FtsY–NG (B) into monolayers is stimulated by phosphatidylglycerol. The increase in surface pressure upon injection of the protein into the subphase was measured as a function of the initial surface pressure. Monolayers were prepared from phospholipid extracts of wild-type inner membrane vesicles of E.coli strain SD12, and of PG–depleted inner membrane vesicles of E.coli strain HDL11.

Fig. 5. Insertion of FtsY and FtsY–NG into a DOPG monolayer (initial surface pressure of 25 mN/m) is influenced by the salt concentration (A) and pH (B) of the subphase buffer. FtsY and FtsY–NG were injected into a subphase of 50 mM Tris–HCl pH 7.5 containing the indicated concentrations of NaCl (A) or 50 mM Na phosphate, 100 mM NaCl at the indicated pH (B).

Fig. 6. Effect of salt concentration on FtsY–induced LUV aggregation (measured as in Figure 2) for different FtsY (FL) concentrations (open circles, 0.1 μM; dotted circles, 0.2 μM; filled circles, 0.4 μM) and constant lipid concentration (100 μM).

Fig. 7. Effect of nucleotides on the insertion of FtsY, FtsY^*449 and FtsY–NG into a DOPG monolayer (initial surface pressure of 25 mN/m). Protein was injected into a subphase buffer of 50 mM Tris–HCl, 100 mM NaCl pH 7.5 containing 2 mM MgCl₂. GTP, GDP or GMP-PNP were added to a final concentration of 2 mM prior to injection of protein.

Fig. 8. FtsY is relatively resistant to protease treatment in the presence of liposomes and GTP. A 4 μg aliquot of FtsY was incubated in the absence (lanes 3–6) or presence (lanes 7–10) of liposomes and 2 mM of nucleotide where indicated. Samples were treated with proteinase K (lanes 3–10) or left untreated (lane 2) at 37°C for 2 min (A) or 20 min (B). Samples were subjected to TCA precipitation, analysed by SDS–PAGE and visualized by Coomassie Blue staining.

Fig. 9. Effect of LUVs on the conformation of FtsY derived from infrared-ATR spectra. (A) Amide I′ spectrum of deuterated films of FtsY–NG (upper solid line) and its deconvolution (lower solid line) obtained with 1.6 resolution enhancement factor and Bessel smoothing. Two major components at 1654 ± 1 cm^–1 (α-helix) and 1636 ± 1 cm^–1 (β-sheet), six minor components at 1682 cm^–1 (β-turn), 1675 cm^–1 (antiparallel β-sheet), 1666 cm^–1 (β-turn), 1625 cm^–1 (β-sheet), 1614 cm^–1 and 1600 cm^–1 (side chains), and a shoulder at 1648 cm^–1 (random coil) are present, assigned according to Byler and Susi (1986) and Tatulian et al. (1995). An initial set of nine Lorentzian components with these positions was used to generate a least-squares fit of the original data. The resulting average secondary structure was 46% α-helix, 39% β-sheet, 10% β-turn and 5% random coil. Errors are typically ± 5% of the determined values. (B) Differential spectrum obtained by subtracting the FtsY–NG spectrum from the normalized FtsY spectrum. (C) Differential spectra obtained by subtracting the soluble form from the normalized lipid-bound form, for either full-length (FL) FtsY (dashed line) or FtsY–NG fragment (solid line).

fig. 10. Effect of LUVs on GTP hydrolysis by FtsY and FtsY–NG. (A) The A–domain acts as a repressor of GTP hydrolysis in FtsY in the absence of lipids. FtsY or FtsY–NG (1 μM) was incubated in hydrolysis buffer (see Materials and methods) at the indicated final concentration of GTP for 22 min at 37°C. (B) FtsY, but not FtsY–NG, is stimulated upon lipid interaction. Initial reaction rates for FtsY and for FtsY–NG are shown as a function of the protein:lipid ratio (the lipid is PG). FtsY (100 nM) and FtsY–NG (1 μM) were incubated at 37°C with 10 μM GTP. Time points were taken at 0, 2, 4 and 10 min. The lipid is PG. (C) Lipid interaction of FtsY seems to affect mainly the v_max. Initial reaction rates have been determined as a function of substrate and lipid concentration and are fitted with the Michaelis–Menten equation (the error of the fit is given in parentheses). FtsY (100 nM) was incubated at 37°C with PG and GTP at the indicated concentrations. The K_m and v_max are given. (D) Stimulation of GTP hydrolysis in FtsY depends on the lipid composition of the LUVs. Conditions are: 1 μM protein, 50 μM GTP, 160 μM LUVs; time is 11 min. The key for (A), (B) and (D) is as indicated in the inset in (A).

fig. 10. Effect of LUVs on GTP hydrolysis by FtsY and FtsY–NG. (A) The A–domain acts as a repressor of GTP hydrolysis in FtsY in the absence of lipids. FtsY or FtsY–NG (1 μM) was incubated in hydrolysis buffer (see Materials and methods) at the indicated final concentration of GTP for 22 min at 37°C. (B) FtsY, but not FtsY–NG, is stimulated upon lipid interaction. Initial reaction rates for FtsY and for FtsY–NG are shown as a function of the protein:lipid ratio (the lipid is PG). FtsY (100 nM) and FtsY–NG (1 μM) were incubated at 37°C with 10 μM GTP. Time points were taken at 0, 2, 4 and 10 min. The lipid is PG. (C) Lipid interaction of FtsY seems to affect mainly the v_max. Initial reaction rates have been determined as a function of substrate and lipid concentration and are fitted with the Michaelis–Menten equation (the error of the fit is given in parentheses). FtsY (100 nM) was incubated at 37°C with PG and GTP at the indicated concentrations. The K_m and v_max are given. (D) Stimulation of GTP hydrolysis in FtsY depends on the lipid composition of the LUVs. Conditions are: 1 μM protein, 50 μM GTP, 160 μM LUVs; time is 11 min. The key for (A), (B) and (D) is as indicated in the inset in (A).

fig. 10. Effect of LUVs on GTP hydrolysis by FtsY and FtsY–NG. (A) The A–domain acts as a repressor of GTP hydrolysis in FtsY in the absence of lipids. FtsY or FtsY–NG (1 μM) was incubated in hydrolysis buffer (see Materials and methods) at the indicated final concentration of GTP for 22 min at 37°C. (B) FtsY, but not FtsY–NG, is stimulated upon lipid interaction. Initial reaction rates for FtsY and for FtsY–NG are shown as a function of the protein:lipid ratio (the lipid is PG). FtsY (100 nM) and FtsY–NG (1 μM) were incubated at 37°C with 10 μM GTP. Time points were taken at 0, 2, 4 and 10 min. The lipid is PG. (C) Lipid interaction of FtsY seems to affect mainly the v_max. Initial reaction rates have been determined as a function of substrate and lipid concentration and are fitted with the Michaelis–Menten equation (the error of the fit is given in parentheses). FtsY (100 nM) was incubated at 37°C with PG and GTP at the indicated concentrations. The K_m and v_max are given. (D) Stimulation of GTP hydrolysis in FtsY depends on the lipid composition of the LUVs. Conditions are: 1 μM protein, 50 μM GTP, 160 μM LUVs; time is 11 min. The key for (A), (B) and (D) is as indicated in the inset in (A).

fig. 10. Effect of LUVs on GTP hydrolysis by FtsY and FtsY–NG. (A) The A–domain acts as a repressor of GTP hydrolysis in FtsY in the absence of lipids. FtsY or FtsY–NG (1 μM) was incubated in hydrolysis buffer (see Materials and methods) at the indicated final concentration of GTP for 22 min at 37°C. (B) FtsY, but not FtsY–NG, is stimulated upon lipid interaction. Initial reaction rates for FtsY and for FtsY–NG are shown as a function of the protein:lipid ratio (the lipid is PG). FtsY (100 nM) and FtsY–NG (1 μM) were incubated at 37°C with 10 μM GTP. Time points were taken at 0, 2, 4 and 10 min. The lipid is PG. (C) Lipid interaction of FtsY seems to affect mainly the v_max. Initial reaction rates have been determined as a function of substrate and lipid concentration and are fitted with the Michaelis–Menten equation (the error of the fit is given in parentheses). FtsY (100 nM) was incubated at 37°C with PG and GTP at the indicated concentrations. The K_m and v_max are given. (D) Stimulation of GTP hydrolysis in FtsY depends on the lipid composition of the LUVs. Conditions are: 1 μM protein, 50 μM GTP, 160 μM LUVs; time is 11 min. The key for (A), (B) and (D) is as indicated in the inset in (A).