Vol 450j29 November 2007jdoi 10 1038 nature06384 REVIEWS Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes Tom A Rapoport1 A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane Most of these proteins are translocated through a protein conducting channel that is formed by a conserved heterotrimeric membrane protein complex the Sec61 or SecY complex Depending on channel binding partners polypeptides are moved by different mechanisms the polypeptide chain is transferred directly into the channel by the translating ribosome a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP and a pushing mechanism is used by the bacterial ATPase SecA Structural genetic and biochemical data show how the channel opens across the membrane releases hydrophobic segments of membrane proteins laterally into lipid and maintains the membrane barrier for small molecules or almost 40 years researchers have been fascinated by the question of how proteins are transported across or are integrated into membranes Pioneering work by G Palade1 demonstrated that in eukaryotic cells secretory proteins cross the endoplasmic reticulum membrane before being transported in vesicles to the plasma membrane The laboratories of G Blobel and C Milstein then discovered that these proteins are directed to the endoplasmic reticulum membrane by signal sequences2 3 A little later signal sequences were also found to direct the translocation of proteins across the bacterial plasma membrane4 5 Genetic experiments identified components required for translocation initially in bacteria and later in yeast6 8 and the establishment of an in vitro system initiated biochemical studies9 All of these achievements set the stage for investigations into the molecular mechanism of translocation which will be the focus of this review Proteins transported across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane include soluble proteins such as those ultimately secreted from the cell or localized to the endoplasmic reticulum lumen and membrane proteins such as those in the plasma membrane or in other organelles of the secretory pathway Soluble proteins cross the membrane completely and usually have amino terminal cleavable signal sequences the major feature of which is a segment of 7 12 hydrophobic amino acids Membrane proteins have different topologies in the lipid bilayer with one or more transmembrane segments composed of about 20 hydrophobic amino acids the hydrophilic regions of these proteins either cross the membrane or remain in the cytosol Both types of proteins are handled by the same machinery within the membrane a protein conducting channel The channel allows soluble polypeptides to cross the membrane and hydrophobic transmembrane segments of membrane proteins to exit laterally into the lipid phase F Structure of the translocation channel The translocation channel is formed from a conserved heterotrimeric membrane protein complex called the Sec61 complex in eukaryotes and the SecY complex in bacteria and archaea for more 1 details see refs 10 and 11 The a and c subunits show significant sequence conservation and both subunits are essential for the function of the channel and for cell viability The b subunits are not essential they are similar in eukaryotes and archaea but show no obvious homology to the corresponding subunit in bacteria The a subunit forms the pore of the channel as initially shown by experiments in which photoreactive probes were systematically placed at different positions of a stalled translocating polypeptide12 all positions predicted to be within the membrane cross linked only to the a subunit of the Sec61 complex indicating that this subunit surrounds the polypeptide chain during its passage across the membrane In addition experiments in which the purified Sec61 SecY complex was reconstituted into proteoliposomes showed that it is the essential membrane component for protein translocation13 15 The channel has an aqueous interior as demonstrated by electrophysiology experiments16 and by measurements of the fluorescence lifetime of probes incorporated into a translocating polypeptide chain17 18 The crystal structure of an archaeal SecY complex provided important insight into how the a subunit forms the channel10 The structure is probably representative of complexes from all species as indicated by sequence conservation and by the similarity to a lowerresolution structure of the Escherichia coli SecY complex determined by electron microscopy from two dimensional crystals19 20 Viewed from the cytosol the channel has a square shape Fig 1a The a subunit is divided into two halves transmembrane segments 1 5 and 6 10 The loop between transmembrane segments 5 and 6 at the back of the a subunit serves as a hinge allowing the a subunit to open at the front the lateral gate The c subunit links the two halves of the a subunit at the back by extending one transmembrane segment diagonally across their interface The b subunit makes contact only with the periphery of the a subunit probably explaining why it is dispensable for the function of the complex The ten helices of the a subunit form an hourglass shaped pore that consists of cytoplasmic and external funnels the tips of which meet about half way across the membrane Fig 1b Whereas the Howard Hughes Medical Institute and Department of Cell Biology Harvard Medical School 240 Longwood Avenue Boston Massachusetts 02115 USA 663 2007 Nature Publishing Group REVIEWS NATUREjVol 450j29 November 2007 cytoplasmic funnel is empty the external funnel is plugged by a short helix The crystal structure therefore represents a closed channel but as will be discussed later biochemical data indicate how it can open and translocate proteins The constriction of the hourglass shaped channel is formed by a ring of six hydrophobic residues that project their side chains radially inward The residues forming this pore ring are amino acids with bulky hydrophobic side chains Ribosome SRP Signal sequence Different modes of translocation The channel alone is a passive pore it must associate with partners that provide a driving force for translocation Depending on the partner there are three known ways in which the channel can function In co translational translocation the main