A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion

Abstract

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane ‘insertase’ YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis.

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

“…The HTL bound to BAM in our EM structure ( Fig. 3a, structure ii) seems to be more open when compared to the previously published low-resolution cryo-EM structure (Botte et al, 2016) (emd3056; Fig. 3a, structure i), and also displays a more prominent periplasmic region.…”

“…The inner-membrane region the HTL is much more open than the previous structure, when visualised alone (Botte et al, 2016). In the new open structure, the locations of the corecomplex SecYEG, SecDF and YidC can be easily distinguished, connected by two bridges from the former (Fig.…”

“…S4a,right ; Table S1), which was selected for analysis by NS-EM. The subsequent single particle analysis of this material (Table S2) revealed a remarkable structure large enough (~ 300 x 250 x 150 Å) to contain both Sec and BAM machineries (Botte et al, 2016;Iadanza et al, 2016), and with a height sufficient to straddle the space between the two membranes (Fig. 2b, Fig.…”

“…3a, structure ii). The 'open' structure, and the 'compact' structure seen before (Botte et al, 2016), may reflect different functional states of the translocon. Presumably, the HTL would be closed when idle in the membrane, and would open to various degrees depending on the associated cytosolic partners (e.g.…”

Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion

“…While SecYEG dimerization does not appear to be driven by specific interactions at Sites 1 or 2, this does not preclude their involvement in other protein-protein interactions. SecYEG can form a complex with the membrane 'insertase' YidC, along with the accessory subcomplexes SecDF and YajC, to form the 'holo-translocon', capable of protein secretion and membrane protein insertion (46,47).The preliminary structure of the holo-translocon (48) predicts that Sites 1 and 2 are proximal to YidC in this complex; indeed, in this context the CGMD data predicts the positioning of the CL head group directly on the C1 region of YidC ( Figure S10; (48, 49)). As with SecYEG alone, CL has been implicated in mediating the interaction of SecA with the HTL (46).…”