The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli

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. coliwhich, 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 ΔμH+. 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.

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Preprotein mature domains contain translocase targeting signals that are essential for secretion

The Journal of Cell Biology ◽

10.1083/jcb.201609022 ◽

2017 ◽

Vol 216 (5) ◽

pp. 1357-1369 ◽

Cited By ~ 38

Author(s):

Katerina E. Chatzi ◽

Marios Frantzeskos Sardis ◽

Alexandra Tsirigotaki ◽

Marina Koukaki ◽

Nikolina Šoštarić ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Secretion ◽

Receptor Site ◽

Secretory Proteins ◽

Signal Peptides ◽

Cytoplasmic Face ◽

Hydrophobic Nature ◽

Mature Domain ◽

Targeting Signals

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion.

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