Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon

Abstract

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

Figure 1

The secY205 mutant is exclusively impaired in the translocation of secretory proteins but not in the integration of polytopic membrane proteins. (a) The polytopic membrane protein mannitol permease (MtlA) and the secretory protein OmpA were synthesized in vitro in a cell-free translation system in the presence of inside-out inner membrane vesicles (INV) that were prepared from wild-type and different secY mutant strains. [³⁵S]methionine-labeled translation products were either directly precipitated with TCA or only after incubation with 0.5 mg/ml proteinase K (PK) for 20 min at 25°C. Indicated are the positions of full-length MtlA, the membrane-protected fragment of MtlA resistant toward proteinase K (MtlA-MPF), the precursor (pOmpA), and the signal sequence–free form of OmpA. The percentage of integration was calculated after quantification of the radioactivity of individual protein bands using a PhosphorImager and calculating the ratio between MtlA-MPF and MtlA. The values obtained were corrected for the loss of Met residues occurring during cleavage by proteinase K. The percentage of translocation equals the ratio of radioactivity present in the proteinase K–resistant bands of pOmpA and OmpA and that recovered from the corresponding bands before proteolytic digestion. The values shown represent the means of at least three independent experiments with the SDs given in parenthesis. (b) Three polytopic inner membrane proteins, SecY, SecY205, a mutated version of SecY, and a lactose permease fusion protein (LacY-Bla) were synthesized in vitro in the presence of wild-type or secY205 INV. Membrane association was analyzed by subfractionation of the reaction mixture on a two-step sucrose gradient. Three fractions were sequentially withdrawn and analyzed on SDS-PAGE: the supernatant (sup), the membrane fraction (mem), and the pellet fraction (pel). Radioactivity present in the individual bands was quantified, and the sum of the three subfractions was each set at 100%.

Figure 2

In contrast to SecE, SecG is not required for the integration of polytopic membrane proteins. (a) MtlA and OmpA were synthesized in vitro as detailed in Fig. 1 in the presence of inner membrane vesicles prepared either from the wild-type, the cold-sensitive secE mutant PS163, the SecE-depletion mutant CM124, or the secG mutant KN553. Integration and translocation were quantified as described in Fig. 1. (b) Integration of SecY and LacY-Bla into INV, derived from the secG KN533 mutant, or the secE mutant CM124, grown in the absence of arabinose, was analyzed as specified in Fig. 1. (c) Immunoblot of wild-type and secE mutant vesicles using rabbit antisera directed against SecY and SecE and an HRP-based enhanced chemiluminescence detection system. (d) Translocation of pLamB was analyzed as specified for pOmpA translocation in Fig. 1.

Figure 2

In contrast to SecE, SecG is not required for the integration of polytopic membrane proteins. (a) MtlA and OmpA were synthesized in vitro as detailed in Fig. 1 in the presence of inner membrane vesicles prepared either from the wild-type, the cold-sensitive secE mutant PS163, the SecE-depletion mutant CM124, or the secG mutant KN553. Integration and translocation were quantified as described in Fig. 1. (b) Integration of SecY and LacY-Bla into INV, derived from the secG KN533 mutant, or the secE mutant CM124, grown in the absence of arabinose, was analyzed as specified in Fig. 1. (c) Immunoblot of wild-type and secE mutant vesicles using rabbit antisera directed against SecY and SecE and an HRP-based enhanced chemiluminescence detection system. (d) Translocation of pLamB was analyzed as specified for pOmpA translocation in Fig. 1.

Figure 2

In contrast to SecE, SecG is not required for the integration of polytopic membrane proteins. (a) MtlA and OmpA were synthesized in vitro as detailed in Fig. 1 in the presence of inner membrane vesicles prepared either from the wild-type, the cold-sensitive secE mutant PS163, the SecE-depletion mutant CM124, or the secG mutant KN553. Integration and translocation were quantified as described in Fig. 1. (b) Integration of SecY and LacY-Bla into INV, derived from the secG KN533 mutant, or the secE mutant CM124, grown in the absence of arabinose, was analyzed as specified in Fig. 1. (c) Immunoblot of wild-type and secE mutant vesicles using rabbit antisera directed against SecY and SecE and an HRP-based enhanced chemiluminescence detection system. (d) Translocation of pLamB was analyzed as specified for pOmpA translocation in Fig. 1.

Figure 2

In contrast to SecE, SecG is not required for the integration of polytopic membrane proteins. (a) MtlA and OmpA were synthesized in vitro as detailed in Fig. 1 in the presence of inner membrane vesicles prepared either from the wild-type, the cold-sensitive secE mutant PS163, the SecE-depletion mutant CM124, or the secG mutant KN553. Integration and translocation were quantified as described in Fig. 1. (b) Integration of SecY and LacY-Bla into INV, derived from the secG KN533 mutant, or the secE mutant CM124, grown in the absence of arabinose, was analyzed as specified in Fig. 1. (c) Immunoblot of wild-type and secE mutant vesicles using rabbit antisera directed against SecY and SecE and an HRP-based enhanced chemiluminescence detection system. (d) Translocation of pLamB was analyzed as specified for pOmpA translocation in Fig. 1.

Figure 3

SecY synthesized in vitro and integrated into INV is biologically active. (a) To reconstitute the secY205 INV in a first step, wild-type SecY and, as a negative control, SecY205 were in vitro synthesized in the presence of secY205 INV (pretreatment). The secY205 INV were isolated and subsequently tested for their ability to support the translocation of in vitro synthesized OmpA. No pretreatment was performed in the control reactions shown in lanes 1–4. Translocation was quantified as specified in Fig. 1. (b) To demonstrate the SRP dependency of the reconstitution process, vesicles were extracted with urea (U-INV) to remove Ffh and FtsY before reconstitution with in vitro synthesized SecY. Readdition of Ffh (SRP) and FtsY (SR) is indicated. After the individual pretreatments, vesicles were isolated and subsequently tested for translocation activity towards OmpA.

Figure 3

SecY synthesized in vitro and integrated into INV is biologically active. (a) To reconstitute the secY205 INV in a first step, wild-type SecY and, as a negative control, SecY205 were in vitro synthesized in the presence of secY205 INV (pretreatment). The secY205 INV were isolated and subsequently tested for their ability to support the translocation of in vitro synthesized OmpA. No pretreatment was performed in the control reactions shown in lanes 1–4. Translocation was quantified as specified in Fig. 1. (b) To demonstrate the SRP dependency of the reconstitution process, vesicles were extracted with urea (U-INV) to remove Ffh and FtsY before reconstitution with in vitro synthesized SecY. Readdition of Ffh (SRP) and FtsY (SR) is indicated. After the individual pretreatments, vesicles were isolated and subsequently tested for translocation activity towards OmpA.