Signal peptides are allosteric activators of the protein translocase

Abstract

Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases.

Fig. 1. Translocase binding and export of proPhoA and its derivatives

a. Equilibrium dissociation constants (Kd) of proPhoA and variants for the translocase. SecA(I304A/L306A) (marked “IL”), or PrlA4/SecYEG were used. x: mutant derivative. “–”u mnjh: no detectable binding, NT: not tested. (n= 3–7). In vivo (b) or in vitro (c and d) translocation of proPhoA and derivatives by wild type or PrlA4/SecYEG translocase. In (b) phosphatase units were converted to protein mass. Proteins visualized by immunostaining (c) were quantified by phosphorimaging (d). The percent of translocated material compared to that of the wild type proPhoA (100%) is indicated above each bar. (n=9).

Fig. 2. Activation energy and stimulation of SecA ATPase under different regimes

a. The activation energy (Ea; KJ/mole; Y axis) of the wild-type translocase and variants was determined in the presence of various preprotein derivatives and in the presence of synthetic signal peptides, as indicated. SecA or SecA(I304A/L306A) and wild type or PrlA4/SecYEG were used. x: mutant derivative. (n=4–15). Mutated residues are indicated in capitals. b. The K_cat values (pmoles Pi/pmol SecA protomer/min) of the translocation ATPase activity of SecA at 37°C divided by those of the corresponding membrane ATPase activity (Fig. S3a) represents the folds of stimulation achieved by the various preprotein segments as indicated. (n=4–15).

Fig. 3. Signal peptides added in trans promote PhoA translocation

a. [³⁵S]-PhoA translocation into wild type SecYEG IMVs driven by proPhoA (wt, L8Q, L14R), M13 procoat or proLamB12(KRR) signal peptides. Lanes 11, 14, 15 are identical to 7, 12, 13 except TX-100 addition prior to proteolysis. Lane 3 (100%): lane 7: 120 (±16) %; lane 9: 7 (±4) %; lane 12: 78 (±10) %; lane 13: 71 (±8) %. (n =3) b. Trapping reaction. Translocase was incubated with [³⁵S]-PhoA and then with nucleotides and/or with signal peptides. Where previously omitted, ATP and/or signal peptide were added. Samples (except lane 1) were chased with non-radiolabelled PhoA at 37°C (except lane 4). Lane 8: after 2min the reaction was chilled (4°C) before translocation resumed. Lane 1 (100%).(n =3) c. proPhoA signal peptide-driven [³⁵S]-PhoA translocation (as in “a”). Lane 2 (100%): lane 4, 5 (±1.1) %; lane 6, 12 (±2.4) %. (n =3)

Fig. 4. Generality and working model of bacterial secretory protein translocation

a. Equilibrium dissociation constants (K_D) of precursor and mature forms of the indicated secretory E. coli proteins for the translocase. b. and c. In vitro translocation reactions contain in b. [³⁵S]-labeled mature forms and synthetic proPhoA signal peptide whereas in c. [³⁵S]-labeled precursor forms of the indicated secretory proteins (as in Fig. 3a). d. Model of post-translational bacterial protein secretion (see text for details). In “I” (bottom), a nascent secretory chain (thick line) carrying a signal peptide (rectangle) is shown to exit the ribosome. A, SecA; Y, SecY. Elongated shapes in “III–V” depict the triggered state.