Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

Abstract

Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the protein-conducting channel SecYEG, and the motor protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to 16 Å. To access the functional size of the SecYEG pore, the precursor of outer membrane protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane protein A conjugates carrying tetraarylmethanes with diameters up to 18 Å, whereas a 29 Å sized molecule blocked the translocation pore. Translocation of the protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of 4-6 Å, the pore accommodates structures of at least 22-24 Å, which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.secretion | Sec-system

Multiple SecA Molecules Drive Protein Translocation across a Single Translocon with SecG Inversion

“…These findings strongly suggest that the translocon undergoes a dynamic structure change caused by PMF. The observation that precursors artificially attached to a large domain can be translocated in the presence of PMF (41,42,55) is also consistent with this idea. As a high concentration of soluble SecA complements the lack of PMF (38), it is likely that cytosolic SecA induces a similar structure change in the translocon.…”

Position-dependent Effects of Polylysine on Sec Protein Transport

“…However, an alternative and more likely possibility is that counter ions within the hydration shell of a precursor protein can migrate with charged residues through the translocation channel. This picture is consistent with recent work that demonstrated the facile transport of 18-Å rigid tags, indicative of a large channel (49), and it agrees with experiments that demonstrated ion and water flux through an open channel (50). Note that a narrow channel that requires dehydration would be energetically expensive.…”

Determination of the Oligomeric State of SecYEG Protein Secretion Channel Complex Using in Vivo Photo- and Disulfide Cross-linking

“…Of course the physiological relevance of either dimer form remains unclear. The front-to-front dimer with the lateral gates facing one another could allow protomers to fuse to form a larger composite channel, which would permit expansion of the channel to dimensions more consistent with two previous estimates (32,33). Such a fused dimer would only be useful for the transit of partially folded, secretory proteins, because it would lack any lateral gate opening for the exit of membrane proteins into the lipid bilayer.…”