How Quality Control Systems AID Sec-Dependent Protein Translocation.

The evolutionarily conserved Sec machinery is responsible for transporting proteins across the cytoplasmic membrane. Protein substrates of the Sec machinery must be in an unfolded conformation in order to be translocated across (or inserted into) the cytoplasmic membrane. In bacteria, the requirement for unfolded proteins is strict: substrate proteins that fold (or misfold) prematurely in the cytoplasm prior to translocation become irreversibly trapped in the cytoplasm. Partially folded Sec substrate proteins and stalled ribosomes containing nascent Sec substrates can also inhibit translocation by blocking (i.e., "jamming") the membrane-embedded Sec machinery. To avoid these issues, bacteria have evolved a complex network of quality control systems to ensure that Sec substrate proteins do not fold in the cytoplasm. This quality control network can be broken into three branches, for which we have defined the acronym "AID": (i) avoidance of cytoplasmic intermediates through cotranslationally channeling newly synthesized Sec substrates to the Sec machinery; (ii) inhibition of folding Sec substrate proteins that transiently reside in the cytoplasm by molecular chaperones and the requirement for posttranslational modifications; (iii) destruction of products that could potentially inhibit translocation. In addition, several stress response pathways help to restore protein-folding homeostasis when environmental conditions that inhibit translocation overcome the AID quality control systems.

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity.

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.