PerspectiveStructural Biology

Surprising News from the PCC

See allHide authors and affiliations

Science  16 Jan 2004:
Vol. 303, Issue 5656, pp. 320-322
DOI: 10.1126/science.1094664

Proteins destined for secretion are targeted by signal sequences to the inner membrane of bacteria or to the endoplasmic reticulum (ER) of eukaryotic cells. At the membrane, these signal sequences initiate the translocation of newly synthesized (nascent) polypeptides across the lipid bilayer. Translocation is envisaged as the threading of the nascent linear polypeptide chain through a protein-conducting channel (PCC)—the SecY complex in bacteria and the evolutionarily conserved Sec61 complex in eukaryotes (1, 2). Newly synthesized membrane proteins are also inserted into these channels, but in contrast to secretory proteins, parts of these proteins—the transmembrane (TM) segments—are not completely translocated across the membrane but are released into the lipid bilayer. The PCC is thought of as a two-way gate with an aqueous pore for transport of a polypeptide across the membrane and a lateral gate for entry of TMs into the lipid bilayer. Much has been learned about the structure and function of the SecY/Sec61 channel [reviewed in (2, 3)]. It is widely accepted—and even reported in textbooks—that the actual pore of the channel is assembled from several SecY/Sec61 complexes. However, the first crystal structure of the PCC from the archaeon Methanococcus jannaschii, recently published by van den Berg et al. in Nature (4), provides strong evidence that a monomeric, not polymeric, SecY complex forms the pore through which proteins are translocated.

The SecY/Sec61 complex is a heterotrimeric membrane protein consisting of an α subunit (SecY in bacteria and Sec61α in mammals), a β subunit (SecG in bacteria and Sec61β in mammals), and a γ subunit (SecE in bacteria and Sec61γ in mammals). The α subunit is thought to span the membrane 10 times, whereas the smaller β and γ subunits each span the membrane once or twice. Site-specific cross-linking of different parts of newly synthesized polypeptides reveals that the hydrophilic pore of the PCC is formed by α subunits. These experiments also show that the hydrophobic segments of signal sequences and TM sequences of the polypeptide move during their insertion from the α subunit of the channel, via an interface, into the lipid bilayer (57).

Low-resolution structures of isolated SecY and Sec61 complexes or of the Sec61 complex bound to a ribosome, obtained by cryo-electron microscopy (cryo-EM), revealed ringlike structures with an outer diameter of about 100 Å, a pore size of 15 to 20 Å, and a calculated mass equivalent to three or four SecY/Sec61 complexes (8, 9). Furthermore, the central cavity in the Sec61 complex was found to align with the exit site of the tunnel in the large ribosomal subunit through which nascent polypeptides are extruded during their synthesis. The observation that large, reactive molecules (20 to 50 Å in size) could gain access to the nascent polypeptide in the channel also provided support for the presence of a large aqueous pore across the membrane (3). The ribosome and chaperone proteins were suggested to seal the pore on the cytoplasmic and lumenal side of the ER membrane, respectively. More recently, the structure of a SecY complex was determined at 8 Å resolution by cryo-EM from two-dimensional crystals (10). The SecY complex appeared to be a dimer with a central cavity measuring 16 Å by 25 Å that was closed on the periplasmic side; a pore was proposed to form between the two monomers (10). These data suggested that the PCC is formed by SecY/Sec61 complex oligomers. However, the results of a biochemical analysis of the oligomeric state of SecY complexes by Yahr and Wickner (11) strongly challenged this view. They concluded that the active form of the PCC associated with its translocation motor SecA is a monomeric SecY complex because (i) formaldehyde cross-linking experiments revealed cross-links between the tetrameric SecY EG subunits but not higher order oligomers of SecY complexes, (ii) a hemagglutinin-tagged SecE did not immunoprecipitate a coexpressed authentic SecE, and (iii) a translocation intermediate was not found associated with multiple copies of the SecY complex. The interpretation of the low-resolution cryo-EM structures clearly did not match the biochemical analyses of Yahr and Wickner, and so the question of whether a functional PCC is formed by a SecY/Sec61 monomer or oligomer remained unanswered.

The x-ray structure reported by van den Berg and colleagues of the Methanococcus SecY complex at 3.2 Å resolution provides strong evidence that a monomeric SecY/Sec61 complex is the functional unit of the PCC (4). For many of us this probably comes as a big surprise. A closer look at the structure reveals that essentially one α subunit forms the functional PCC (see the figure, A). The dimensions of this channel are not that different from those of membrane transporter proteins such as the lactose permease (12). The α subunit contains 10 α-helical TM segments arranged like a clamp (see the figure, A). The two halves of the clamp comprise TM segments 1 to 5 and 6 to 10, which are related by a two-fold symmetry. TM segments 2 and 7 of the α subunit are positioned at the opening of the clamp, presumably where TM segments of membrane proteins are released into the lipid bilayer (13). The small β and γ subunits are attached to the periphery of the α subunit. The pore of the proposed channel is filled by a short distorted helix (TM2a) extending halfway to the center of the membrane. TM2a may act as a plug that must be pushed aside by the incoming signal peptide of the nascent polypeptide chain. Data from bacteria containing signal sequence suppressor mutations support this interpretation (14). When the plug is removed, a constriction called the “pore ring” maintains the membrane barrier. The pore ring is formed by six conserved hydrophobic amino acid residues that seal the channel about halfway in the membrane. This ring is proposed to widen, probably by shifts in the helices forming the channel, to allow the passage of an extended polypeptide chain or even an α helix. Conserved glycine residues in loop regions that fold into the membrane are proposed to act as hinges for these rearrangements. The structure provides an elegant answer to the long-debated question of how the membrane barrier is maintained during the passage of a hydrophilic polypeptide across the lipid bilayer. The channel itself provides structural elements that seal the pore. The cavity seen in the initial cryo-EM structures certainly does not match the pore of a monomeric PCC (8, 9). The central cavity must now be interpreted as a lipid-filled space between a complex of four SecY/Sec61 monomers. Recent EM structures at higher resolution are consistent with this interpretation as they no longer show a central cavity (15, 16). The size and mass of this PCC oligomeric complex attached to the ribosome (8, 9) suggested that it consists of four Sec61 heterotrimers. According to the new structural data of van den Berg et al., this complex would have four channels (4). Van den Berg et al. argue convincingly that the channels may be arranged as two back-to-back dimers with the lateral exits facing toward the lipid bilayer (see the figure, B).

Crossing membranes.

(A) A bacterial SecY monomer is shown in a top view as a ribbon model [from (4)] (left), and redrawn as a contour model (blue) (right). The contour model shows a polypeptide chain (orange) in the proposed pore during translocation and the signal sequence (red) of the polypeptide in the lateral gate and then released (black arrow) to the lipid bilayer. (B) (Left) SecA (green) mediates translocation of a newly synthesized secretory protein (orange) by the monomeric SecY complex (blue) across the bacterial plasma membrane. (Right) The eukaryotic PCC, Sec61 (blue), associated with a ribosome (green) is shown. Top and side views of the SecY complex were redrawn from (4). The ribosome with an attached protein-conducting channel in the side and top view was redrawn from (9).

What would be the advantage of having four PCC channels at the ribosome? One answer may be that secretory proteins and different types of membrane proteins are inserted into different PCC channels. It is conceivable that, for instance, cleavable signal sequences and type I and type II signal anchor sequences are directed to different channels for amino-and carboxyl-terminal translocation, respectively. The discriminating feature in the signal sequences of these different proteins could be the charged amino acid residues that flank the hydrophobic core of a signal sequence on either the amino-or carboxyl-terminal end. Furthermore, membrane proteins that span the membrane multiple times and that have large cytoplasmic domains might be inserted into the membrane by the sequential use of neighboring channels. Properties of newly synthesized polypeptide chains might be “sensed” in the ribosomal exit tunnel and then would be transmitted to the PCC and associated proteins. This type of “sensing” has been observed for transmembrane sequences of newly synthesized membrane proteins (3). In addition, enzymes involved in the modification of newly formed polypeptides (signal peptidase, specific chaperones, oligosaccharyl-transferase) might be selectively recruited by the ribosome to a particular channel. The ribosome then would become a sorting station for signal sequences and different types of signal anchor sequences, thereby actively participating in the insertion of the nascent polypeptides into the PCC.

The first x-ray structure of a SecY complex has drastically changed our view of the PCC, providing us with an understanding of how it opens and closes. However, the new structure also raises a number of intriguing questions, including the functional consequences of having four SecY/Sec61 monomers with four separate translocation channels forming the PCC.


  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
View Abstract

Stay Connected to Science

Navigate This Article