Projection structure and oligomeric properties of a bacterial core protein translocase - PubMed (original) (raw)

Projection structure and oligomeric properties of a bacterial core protein translocase

I Collinson et al. EMBO J. 2001.

Abstract

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

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Figures

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Fig. 1. Purification of SecYEG. SDS–PAGE of fractions during the purification of SecYEG. The detergent extract from membranes is shown on the left adjacent to the molecular weight markers (94, 67, 42, 30, 20 and 12 kDa). The unbound protein from the nickel column is next to the right and is clearly depleted in SecY and SecE. The partially pure SecYEG eluted from the same column and the eluted fractions across the main peak from the gel filtration column are also shown. The star indicates the migration position for contaminants arising from a cleavage in SecY.

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Fig. 2. Subunit composition of the SecYEG complex. (A) Immuno precipitation using SecY or SecG antibodies. A 5 µl aliquot (10%) of the total bound material was applied directly to the gel. Lanes 1 and 2 denote the material precipitated by SecY and SecG antibodies, respectively. Lane 3 is a sample of purified SecYEG for reference. The molecular weight markers are the same as those shown in Figure 1. The positions of SecY, E and G are indicated. (B) SDS–PAGE analysis of the crystalline SecYEG specimen (lane 4).

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Fig. 3. Activity measurement of purified SecYEG. (A) ATPase assays were with either SecA alone, SecA with liposomes containing SecYEG purified in C12E9 and proOmpA, or SecA with liposomes containing SecYEG purified in dodecyl maltoside (dod-malt) and proOmpA. (B) proOmpA translocation into proteoliposomes was tested using membrane-reconstituted vesicles from dodecyl maltoside (proteo DDM; lanes 4 and 5, with and without ATP, respectively) or C12E9 (proteo C12E9, lanes 6 and 7) purified SecHisEYG. Lanes 2 and 3 represent translocation experiments using SecHisEYG-enriched inverted inner membrane vesicles (IMVs HisEYG) for comparison. Lane 1 includes proOmpA (pOA, 10% of the total reaction was applied) for reference. Reactions were incubated for 15 min at 37°C with or without ATP.

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Fig. 4. Sedimentation equilibrium analysis of SecYEG. (A) Experimental absorbance versus radius data (open circles), best fit to the data assuming a monomer–dimer–tetramer model of self-association (—), and calculated local contributions of SecYEG monomers (open squares), dimers (filled circles) and tetramers (open triangles) to A(r). Initial protein concentration: 4.8 µM (_A_1.2 cm230 nm = 0.36). Rotor speed: 13 000 r.p.m. Rotor temperature: 4°C. (B) Statistical accuracy of the calculated relative absorbance contributions _Â_i, of the different SecYEG oligomers [obtained from the _A_i(_r_o) values by integrating over the sample volume]: changes in the sum of the squared residuals, σ, of fits to the data from (A), which result from one non-optimal absorbance parameter (Schuck, 1994; Schuck et al., 1995). The minima of the curves correspond to the best fit figures for _Â_i. The nomenclature is the same as in (A). (C) Dependency of the local weight average molar mass, _M_w,app., of SecYEG on the local protein concentration for two samples with loading concentrations of 0.14 mg/ml (- – -) and 0.28 mg/ml (—). Rotor speed, 11 000 r.p.m.; rotor temperature, 4°C. (D) Oligomer distribution of the mutant protein SecYprlA4EG. The graph corresponds to that shown in (B) for the wild-type protein. Initial protein concentration: 4.8 µM. Rotor speed, 15 000 r.p.m.; rotor temperature, 4°C.

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Fig. 4. Sedimentation equilibrium analysis of SecYEG. (A) Experimental absorbance versus radius data (open circles), best fit to the data assuming a monomer–dimer–tetramer model of self-association (—), and calculated local contributions of SecYEG monomers (open squares), dimers (filled circles) and tetramers (open triangles) to A(r). Initial protein concentration: 4.8 µM (_A_1.2 cm230 nm = 0.36). Rotor speed: 13 000 r.p.m. Rotor temperature: 4°C. (B) Statistical accuracy of the calculated relative absorbance contributions _Â_i, of the different SecYEG oligomers [obtained from the _A_i(_r_o) values by integrating over the sample volume]: changes in the sum of the squared residuals, σ, of fits to the data from (A), which result from one non-optimal absorbance parameter (Schuck, 1994; Schuck et al., 1995). The minima of the curves correspond to the best fit figures for _Â_i. The nomenclature is the same as in (A). (C) Dependency of the local weight average molar mass, _M_w,app., of SecYEG on the local protein concentration for two samples with loading concentrations of 0.14 mg/ml (- – -) and 0.28 mg/ml (—). Rotor speed, 11 000 r.p.m.; rotor temperature, 4°C. (D) Oligomer distribution of the mutant protein SecYprlA4EG. The graph corresponds to that shown in (B) for the wild-type protein. Initial protein concentration: 4.8 µM. Rotor speed, 15 000 r.p.m.; rotor temperature, 4°C.

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Fig. 5. Electron micrograph of a negatively stained two-dimensional crystal of SecYEG. Crystals stained by uranyl acetate were visualized using a Philips CM12 electron microscope. The tubular vesicle shown is flattened to the carbon surface and contains a two-dimensional crystal on each face. Bar, 0.5 µm.

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Fig. 6. Crystallographic data of SecYEG embedded in trehalose. (A) Computer-generated Fourier transform of an image of a SecYEG crystal. Each symbol represents a reflection, and the size of the box and the number within it (IQ values) relate to the quality of the data: a large box and a low number reflect a high signal to noise reflection (Henderson et al., 1986, 1990). The program MMBOXA was used to generate the data (Crowther et al., 1996). The rings show the position where the CTF is zero, and the axes refer to the reciprocal lattice vectors. The edge of the plot corresponds to a resolution of 6 Å. (B) Phase error of unique Fourier components plotted as a function of resolution. The size of the box indicates the error associated with each measurement (1 <8°, 2 <14°, 3 <20°, 4 <30°, 5 <40°, 6 <50°, 7 <70°, 8 <90°, where 90° is random). Values from 1 to 4 are shown as numbers, and decreasing box size indicates higher values. In this representation, measurements for reflections that are forbidden for P121 symmetry ([0, k (odd)]) were not removed, but were omitted upon imposing the P121 symmetry.

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Fig. 7. Projection structure of SecYEG. Projection maps calculated from merged amplitudes and phases from 12 independent lattices of crystals embedded in trehalose. (A) Map drawn to 9 Å in P1 (no symmetry imposed). (B) With P121 symmetry imposed. An isotropic temperature factor of –600 Å2 was applied in (A) and (B) to compensate partially for the resolution-dependent degradation of image-derived amplitudes (Unger et al., 1997a). (C) Map at 9 Å resolution with imposed P121 symmetry without temperature factor adjustments. (D) Map at 15 Å resolution with P121 symmetry imposed. In all cases, four unit cells are shown, and one unit cell (containing two symmetry-related asymmetric units) is boxed. The solid lines indicate densities above the mean. Scale bar, 20 Å.

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Fig. 8. Proposed molecular boundary of SecYEG. Projection map of SecYEG (Figure 7B and D) with the molecular boundaries outlined. The upper panel corresponds to the 9 Å projection structure (Figure 7B) and the lower to 15 Å (Figure 7D).

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