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. 2014 Feb 6;506(7486):102-6.
doi: 10.1038/nature12720. Epub 2013 Oct 23.

Structure of the SecY channel during initiation of protein translocation

Eunyong Park  1 , Jean-François Ménétret  2 , James C Gumbart  3 , Steven J Ludtke  4 , Weikai Li  1 , Andrew Whynot  1 , Tom A Rapoport  1 , Christopher W Akey  2
Affiliations

Structure of the SecY channel during initiation of protein translocation

Eunyong Park et al. Nature. .

Abstract

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryo-electron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.

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Figures

Figure 1
Figure 1. Structures of non-translating ribosome–channel complexes
a, Density map for the M. jannaschii complex. Models for ribosomal RNA and proteins of the small and large ribosomal subunits (ssu and lsu; in gold and blue, respectively) and of the SecY complex (in red) were docked into the map. b, Fit of the M. jannaschii SecY complex into the segmented density map, as viewed from the cytoplasm (top view) and from the side. The N- and C-terminal halves of SecY are in light blue and red, respectively. SecE is in dark blue and Secβ in brown. c, Comparison between the crystal structure of an M. jannaschii SecY complex (grey) and the EM structure (in color), as viewed facing the lateral gate (front view). d-e, As in a and b, but for the E. coli complex. SecG, the bacterial equivalent of Secβ, is in brown. f, A model for the E. coli channel in a front view.
Figure 2
Figure 2. Purification of a ribosome–nascent chain–channel complex
a, The complex was generated in living E. coli cells by expressing a nascent chain (NC) of 100 amino acids with a signal sequence and SecM-stalling sequence. The NC also contains a Myc-tag. A cysteine at position 19 of the NC (19C) was disulfide-crosslinked to a cysteine in the plug of SecY (68C). b, Coomassie-stained SDS-gel of the ribosome-NC (RNC)–channel complex (lane 1). The red arrow indicates the crosslinked product of SecY and the NC-tRNA adduct. This band disappears after treatment with β-mercaptoethanol (β-ME) or RNaseA (lanes 2 and 3). Ribosomal proteins (including S1) and the fusion between SecE and SecG are indicated.
Figure 3
Figure 3. Structure of the active SecY channel
a, Structure of the E. coli RNC–SecY channel complex, with large and small ribosomal subunits in blue and gold, respectively, the SecY complex in red, and ribosomal protein S1 in tan. b, Front and side views of the channel fit into the segmented density map (grey). The nascent chain was omitted for clarity. The N-terminal half of SecY is in light blue, the C-terminal half in red, SecE in dark blue, and SecG in brown. c, Comparison of front views of the closed and open E. coli SecY channels with the approximate position of the membrane indicated by solid horizontal lines. The N-terminal half of SecY is in light blue, the C-terminal half in red, SecE in dark blue, SecG in brown, and the plug in yellow. Some movements during channel opening are indicated, such as the rotation and tilting of the N-terminal half of SecY, the tilting of SecE, and the movement of helix 8b. Labels for helices 2b and 7 are placed at the same position in the closed and open channel. Pore residues forming the constriction in the closed channel are indicated with grey balls and sticks. d, Connections of the ribosome with the 8/9 loop of SecY and the cytoplasmic helix of SecE in the closed and open channels (upper and lower panels, respectively). Note the large movement of helix 8b towards the membrane. e, As in c, but viewed from the top.
Figure 4
Figure 4. Path of the nascent chain
a, Density (in light gold) and model (green line) for the nascent chain (NC) in the RNC-channel complex. The P-site tRNA is in brown, the ribosome in grey, and the channel in blue. The right upper panel shows the entire RNC-channel complex from the same viewing angle. The right lower panel shows the density and model for the NC, with ribosome and channel omitted. The asterisk indicates density for an alternative orientation of the NC loop on the cytoplasmic side of the channel (see also d). b, Side view of the signal sequence (ss) helix in the lateral gate. Density for the NC on the cytoplasmic surface was removed for clarity. c, As in b, but viewed from the top along the axis of the signal sequence helix. d, As in c, but from a slightly different angle of view with NC density on the cytoplasmic surface included. e, As in d, but without the density map.
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References

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