Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

Abstract

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.

The action of cardiolipin on the bacterial translocon

“…Of the bilayer constituents, the response seems to be specific to CL and to a lesser degree PG. The results explain a number of observations specifying the requirement of acidic phospholipids in protein translocation (1)(2)(3)(4)(5)23). The inherent symmetry of CL, with two glycerol-linked PGs, might be utilized in the interaction with twin copies of SecYEG.…”

“…In keeping with this, the bacterial protein translocation reaction, driven by the ATPase SecA through the conserved SecYEG complex, has a known requirement for acidic phospholipids (1)(2)(3)(4)(5).…”

“…The influence of the latter acidic pair on the SecA structure and activity has been well documented: SecA dimers form monomers upon contact with acidic phospholipids (5,7,8), as they do when bound to SecYEG (9). The association and activation of SecA at the cytosolic surface of the membrane requires constituent PG (1,3).…”

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

“…We previously described the incorporation of the SecY monomer into discs using the membrane scaffold protein 1 (MSP1, referred to as Nd-Y) (23). We also reported that SecA and Syd formed a tight complex with the Nd-Y particles (23,26) (Fig.…”

“…Protein translocation taking place at one SecY copy might be facilitated by the second, or translocation may strictly depend on the dimer. The question is further complicated by the dynamic dimeric state of SecA and whether one or two SecA molecules bind to the channel (22)(23)(24)(25).…”