A Dual Function for SecA in the Assembly of Single Spanning Membrane Proteins in Escherichia coli

Abstract

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particledependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle-and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecAindependent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.Membrane protein assembly in both eukaryotes and prokaryotes is initiated by the cotranslational targeting of ribosome-associated nascent chains (RNCs) 3 to the Sec translocons in the endoplasmic reticulum or the bacterial cytoplasmic membrane. This requires binding of the signal recognition particle (SRP) to the signal anchor sequence of a membrane protein when it emerges from the ribosomal exit tunnel and the subsequent interaction of the SRP-RNC complex with the membrane-attached SRP receptor (SR) (1, 2). The subsequent transfer of the RNC to the Sec translocon is probably favored by the proposed close vicinity of the SR to the Sec translocon (3). A direct interaction between FtsY, the bacterial SR, and SecY has recently been demonstrated in Escherichia coli (4). In contrast to the eukaryotic SRP, which targets both membrane and secretory proteins, the bacterial SRP is predominantly engaged in membrane protein targeting. The vast majority of bacterial secretory proteins, i.e. proteins that are destined to reach the periplasmic space or the outer membrane, are post-translationally targeted by the bacteria-specific SecA/SecB pathway (5). In this pathway, SecB functions as a secretion-specific chaperone for most secretory proteins, whereas SecA is proposed to translocate the preprotein in a stepwise translocation of stretches of about 30 amino acids (6 -8).The translocation of large luminal domains in eukaryotic membrane proteins does not depend on cytosolic proteins other than SRP (3). This is different for bacterial membrane prote...

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

“…In vivo studies showed that SRP targets IM proteins to the SecYEGDF translocon for insertion (23,60), while in vitro cross-linking of nascent IM proteins revealed contacts with Ffh, Fts, SecY, and SecA (43,52). SecA is required for the insertion of IM proteins with periplasmic loops longer than 30 amino acids (1,19,43), while single-spanning IM proteins such as PulG always require SecA, regardless of the length of their periplasmic domain (15). Consistent with this, SecA depletion almost completely abolished PrePulG* processing.…”

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