In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways ofEscherichia coli

Abstract

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ⌬H ϩ . In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

“…6). Multiple factors render this assay an optimal choice for testing insights from biophysical studies of SRP in a complete targeting reaction: (i) Bacterial SRP and FtsY mediate pPL targeting as efficiently as their mammalian homologs despite the heterologous nature of this assay (61), highlighting the remarkable conservation of the SRP pathway; (ii) wheat germ extract is devoid of endogenous SRP and potential SRP regulators that may serve redundant functions as TF, which allows the effect of TF to be most readily unmasked; (iii) the microsomal membrane has much higher cotranslational translocation activity than bacterial membrane vesicles (62)(63)(64), so that the output of the assay is not limited by the downstream translocation steps; and (iv) rapid cleavage of signal sequences by signal peptidase allows both the premature and mature proteins to be quantified, providing the most accurate quantification of targeting efficiency; analogous readouts are not available with bacterial membrane vesicles. Finally, the Sec translocon has a more relaxed specificity than SRP and translocates both cotranslational and posttranslationally targeted substrates (65); thus, the overall assay reports on the more stringent substrate selection by the SRP pathway.…”

Is Ffh required for export of secretory proteins?

“…This genetic screening strategy suggested that only membrane proteins serve as SRP substrates. Furthermore, in vitro studies with puri¢ed components revealed that membrane proteins required SRP components, but not SecA/SecB, for membrane integration, whereas secretory proteins required SecA/SecB, but not SRP components, for translocation [5]. These latter studies suggest that most E. coli secretory proteins can utilize the SRP-independent pathway with high e¤ciency.…”

Glycolipozyme membrane protein integrase (MPIase): recent data

“…Efficient integration into membranes was observed, even if the SecYEG translocon was absent, when MtlA was in vitro synthesized with liposomes composed of Escherichia coli phospholipids (7). This observation is not compatible at all with the in vitro results obtained using INV prepared from mutant strains (12,13); therefore, it must be an artifact owing to the artificial membrane system (7). Nonetheless, it should reflect membrane integration because it was stimulated by SRP/SR, and resistant against urea and high-salt extraction.…”

“…In the case of MtlA (mannitol permease) with six TMs, an MPF of ∼30 kDa is generated upon membrane integration (10,11). The in vitro system involving INV prepared from mutant strains demonstrated that MtlA is targeted to a membrane by SRP and SR, followed by integration into the membrane at the SecYEG translocon; that is, it is Sec dependent (12,13). Efficient integration into membranes was observed, even if the SecYEG translocon was absent, when MtlA was in vitro synthesized with liposomes composed of Escherichia coli phospholipids (7).…”

“…Nonetheless, it should reflect membrane integration because it was stimulated by SRP/SR, and resistant against urea and high-salt extraction. Thus, MtlA was proven to be spontaneously integrated into liposomes (7), while such spontaneous integration is strictly blocked in INV (12,13). These findings led us to the hypothesis that membrane components such as minor lipids and membrane proteins in INV inhibited the spontaneous integration.…”