蛋白传导通道的结构

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X-ray structure of a protein-conducting channel

BERT VAN DEN BERG1,*, WILLIAM M. CLEMONS JR1,*, IAN COLLINSON2, YORGO MODIS3, ENNO HARTMANN4, STEPHEN C. HARRISON3 & TOM A. RAPOPORT1

Nature 427, 36 - 44 (01 January 2004); doi:10.1038/nature02218


A decisive step in the biosynthesis of many secretory and plasma-membrane proteins is their transport across the endoplasmic reticulum (ER) membrane in eukaryotes or across the cytoplasmic membrane in prokaryotes (for a review, see ref. 1). These polypeptides are first targeted to the membrane by hydrophobic amino-acid sequences, which are either cleavable signal sequences or transmembrane segments (TM) of membrane proteins. Soluble proteins, such as those destined for secretion, are subsequently transported across the membrane through a protein-conducting channel with a hydrophilic interior2, 3. In the case of membrane proteins, when a hydrophobic TM arrives in the channel, it is released through an opening in the channel wall into the surrounding lipid phase. The capacity of the channel to open laterally towards the lipid and the wide variety of substrates that it must transport distinguish it from many other channels.
An evolutionarily conserved heterotrimeric complex of membrane proteins, called the Sec61 complex in eukaryotes and the SecY complex in eubacteria and archaea, forms the channel (for a review, see ref. 4). The -subunits (Sec61 in mammals, Sec61p in Saccharomyces cerevisiae, SecY in bacteria and archaea) and -subunits (Sec61 in mammals, Sss1p in S. cerevisiae, SecE in bacteria and archaea) show significant sequence conservation (see Supplementary Fig. S1). Both subunits are required for cell viability in S. cerevisiae and Escherichia coli. The -subunits (Sec61 in mammals, Sbh in S. cerevisiae, Sec in archaea) are not essential for cell viability in these organisms; they are similar in eukaryotes and archaea, but show no obvious homology to the corresponding SecG subunits in bacteria. The -subunit forms the channel pore, and it is the crosslinking partner of polypeptide chains passing through the membrane5. Reconstitution experiments have shown that the Sec61/SecY complex is the essential membrane component for protein translocation6, 7.

The channel itself is a passive conduit for polypeptides and must therefore associate with other components that provide a driving force1. In co-translational translocation, the major partner is the ribosome. The elongating polypeptide chain moves directly from the ribosome into the associated membrane channel. The energy for translocation comes from GTP hydrolysis during translation. Many (or perhaps all) cells also have post-translational translocation, in which polypeptides are completed in the cytosol and then transported across the membrane. In yeast (and probably in all eukaryotes), the post-translational translocation partners are another membrane protein complex (the tetrameric Sec62/63p complex) and the lumenal protein BiP, a member of the Hsp70 family of ATPases8, 9. BiP promotes translocation by acting as a molecular ratchet, preventing the polypeptide chain from sliding back into the cytosol10. In the eubacterial post-translational pathway, the cytosolic ATPase SecA pushes polypeptides through the channel11. In addition, an electrochemical gradient across the membrane stimulates translocation in vitro and is essential in vivo12. Archaea lack SecA and the Sec62/63p complex, and it is unclear how they perform post-translational translocation13. Despite the differences between the pathways, most mechanistic aspects of translocation that relate to the channel itself are probably similar. Specifically, in all cases the channel partner—either the ribosome, the Sec62/63p complex or SecA—binds first, and the signal sequence or TM of a translocation substrate associates with the channel subsequently, priming it for polypeptide translocation.

An understanding of the mechanisms that underlie protein translocation requires detailed structural information. Low-resolution structures have been obtained by single-particle electron microscopy (EM) of either the isolated Sec61/SecY complex or the Sec61 complex bound to the ribosome14-18. A recent structure of the E. coli SecY complex, derived from electron cryo-microscopy of two-dimensional (2D) crystals in a phospholipid bilayer, indicated the expected number of TM helices, but the resolution (about 8