![]() ![]() The presence of the translocon subunits in the equivalent region of the native gel from HTL alone ( Figure 1a,b, region 1) is due to the HTL-dimer, which over laps with this area of the gel. The results clearly show the existence of a ∼400 kDa complex containing both the HTL subunits and SurA ( Figure 1a,b, regions 4 and 5). A kinetic analysis of the maturation and folding of the OMP LamB showed that surA mutants delay signal sequence cleavage consistent with an interaction during, or very shortly after, translocation through the inner membrane. Thus, it is almost self-evident that SurA needs to interact with OMPs, prior to their release from the Sec machinery, to minimise the potential for aggregation in the periplasm. Critically, the activity of SurA in inter-membrane transport depends on an interaction prior to aggregation, as unlike the Skp, it lacks the ability to recover aggregated substrates. SurA is thought to be the dominant chaperone for outer-membrane delivery due to its known affinity for both the hydrophobic motifs characteristic of OMPs, and the BAM complex in the outer membrane. Unlike cytosolic quality control factors, they must somehow operate in the absence of ATP, perhaps by virtue of their structural plasticity. During their journey through this crowded environment, periplasmic chaperones such as SurA, Skp, PpiD and the protease DegP are recruited to help prevent and resolve aggregation. Similar to the trafficking process to and across the inner-membrane, OMPs must remain unfolded during subsequent passage through the periplasm until they reach the outer-membrane. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane. Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG–SecDF–YidC - aka the holo-translocon (HTL). However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). ![]() Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein–channel. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. ![]() Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Access content during the Covid-19 pandemic.
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