The bacterial translocon SecYEG opens upon ribosome binding

Abstract

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist.

Keywords: Channel Gating; Membrane Bilayer; Membrane Biophysics; Membrane Reconstitution; Permeability; Protein Translocation.

FIGURE 1.

Single channel activity of the wild-type SecY channel induced by ribosome binding. Left panel, single channel traces at different voltages. Right panel, the corresponding amplitude histograms.

FIGURE 2.

Channel activation by isolated ribosomal subunits. A, the isolated 30 S subunit was unable to activate wild-type SecY. B, channel activity was observed only upon subsequent or sole addition of isolated 50 S subunits to the same membrane. Representative single channel traces at different voltages are shown. C, amplitude histograms corresponding to the recordings in B.

FIGURE 3.

Point mutation R357E does not inhibit ribosome binding. Left panel, ribosome-induced single channel activity observed with the double mutant SecY complex (F67C/R357E). Right panel, the corresponding amplitude histograms.

FIGURE 4.

Partial inhibition of ribosome binding by ATA. Left panel, the upper trace shows the simultaneous openings of five wild-type translocation channels. The addition of ATA reduced the number of open channels to two (lower trace), indicating that ATA partially inhibits ribosome binding. Right panel, the corresponding amplitude histograms.

FIGURE 5.

Current-voltage characteristics of single SecY complexes activated by whole ribosomes and the 50 S ribosomal subunit. The channel amplitude is highly reproducible. Error bars indicate S.D. and are not shown if they were smaller than the symbol. The slope of the linear regression corresponds to a single channel conductance (g) of ∼532 picosiemens.

FIGURE 6.

A, probability of SecYEG to open upon ribosome binding. A suspension of proteoliposomes containing the ATTO 488-labeled SecY(A204C) complex was measured by fluorescence correlation spectroscopy. The autocorrelation function (G(τ)) indicated that, on average, 0.15 vesicles were in the confocal volume. Dissolving the vesicles by detergent increased the particle number to 0.5. The 0.15/0.5 ratio indicated that every vesicle contained approximately three copies of the SecY complex. B, in the presence of ribosomes, fusion of these vesicles with preformed planar bilayers resulted in channel activity. The initial current jump indicates the fusion of a single vesicle, which contained four copies of the SecY complex, as derived from the ratio of the initial current jump to the single channel amplitude.