The SecY complex forms a channel capable of ionic discrimination

Abstract

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring-structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion-conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring-structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.

Figure 1

Ionic conductance of the SecY complex. (A) Inner membrane vesicles (IMVs) with the wild-type (SecY^WT) complex were diluted in buffer A (50 mM Tris–SO₄ pH 7.9, 25 mM K₂SO₄, 10 mM MgSO₄, 0.2 mg/ml BSA) in the presence of oxonol VI (2 μM). The membrane potential Δψ, inside positive, was generated with NADH (5 mM). The stability of Δψ was then challenged using various potassium salts (each 5 mM) for approximately 1 min and then dissipated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 1 μM). The graph (left panel) represents the percentage of Δψ that has been dissipated 10 s after the addition of salt. The traces (right panel) are examples of fluorescence recordings. (B,C) IMVs with the indicated SecY mutant complex were analysed as described above. The SecY mutants that were tested are (B) SecY^PrlA4—mutations I408N and F286Y in SecY (recordings shown in the right panel); (C, left panel) SecY^Δ33—deletion of 33 amino-acid residues in the SecY plug domain; and (C, right panel) SecY^67CC + bismaleimidoethane (BMOE)—SecY complex with cysteine residues at position 67 of SecY and position 120 of SecE in a crosslinked state. Error bars indicate the standard deviation between three experiments.

Figure 2

The SecY pore mutants that are the most active in protein translocation are also the most leaky, but for only monovalent anions. (A) The ionic conductance of inner membrane vesicles with the indicated SecY mutant complex was analysed as described in Fig 1A. (B) The in vivo protein translocation activity of the mutants was assayed using the alkaline phosphatase 14R as a reporter. (C) The in vitro protein translocation activity of the mutants was assayed using ¹²⁵I-labelled proOmpA (pOmpA). The quantification was carried out by densitometry scanning using Image J (build 1.40 g). WT, wild type.

Figure 3

The ionic specificity of the SecY complex is a conserved characteristic. A membrane potential Δψ was generated and measured as described in Fig 1A, but using inner membrane vesicles with the Methanococcus jannaschii SecYE complex, either (A) wild-type or (B) carrying a prlA4-like mutation L406N. The graphs represent the percentage of Δψ that has been dissipated 10 s after the addition of salt.

Figure 4

The Cl⁻-conductive SecY mutant channels are impermeable to protons. (A) The inner membrane vesicles (IMVs) with the indicated SecY mutant were incubated in buffer A in the presence of 9-amino-6-chloro-2-methoxyacridine (ACMA; 4 μM). A gradient of protons, inside acidic, was generated with 1 mM NADH and abolished with 1 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The injection of 5 mM KCl causes a Cl⁻ influx in the mutant allowing for further acidification inside the vesicles. (B) IMVs bearing the SecY^Δ33 complex were prepared as described in (A), but mixed with limiting amounts of NADH (final concentration as indicated). (C) The experiment in (B) was repeated using IMVs with the indicated SecY mutants. For each mutant listed, 100% corresponds to the ΔpH that was generated with 1 mM NADH.

Figure 5

Ionic conductance during protein translocation. (A) 40 μg of inner membrane vesicles (IMVs) with the SecY^WT complex was incubated in 150 μl of buffer B (containing 25 mM KCl) in the presence of bovine serum albumin (30 μg), SecA (3 μg) and the protein substrate proOmpA (pOmpA; 3 μg). Δψ was generated with 5 mM NADH and protein translocation was initiated with 1 mM ATP. (B) IMVs with the wild-type SecY complex (40 μg) were tested as described above, but in buffer A in which K₂SO₄ had been replaced by 25 mM of the indicated salt. The graph represents the percentage of Δψ that was dissipated 10 s after the addition of ATP. (C) SecY^WT IMVs (40 μg) were tested as described in (A), but the translocation reaction was carried out under the following conditions: 1 mM ATPγS; 3 μg of the translocation-incompetent LpK or OmpA; and 40 μg of IMVs with the indicated SecY mutant, at 4–25°C. The graph was generated as described in (B). (D,E) IMVs enriched for the SecYEG or SecYE complexes were tested as in (A) but using 4 μM 9-amino-6-chloro-2-methoxyacridine (ACMA) to follow the variations of ΔpH. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; WT, wild type.