A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

The conserved protein-conducting channel, referred to as the Sec61 channel in eukaryotes or the SecY channel in eubacteria and archaea, translocates proteins across cellular membranes and integrates proteins containing hydrophobic transmembrane segments into lipid bilayers. Structural studies illustrate how the protein-conducting channel accomplishes these tasks. Three different mechanisms, each requiring a different set of channel binding partners, are employed to move polypeptide substrates: The ribosome feeds the polypeptide chain directly into the channel, a ratcheting mechanism is used by the eukaryotic endoplasmic reticulum chaperone BiP, and a pushing mechanism is utilized by the bacterial ATPase SecA. We review these translocation mechanisms, relating biochemical and genetic observations to the structures of the protein-conducting channel and its binding partners.

Gram-negative bacteria and their complex cell envelope comprising an outer and inner membrane are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular penetration of small molecules, including nutrients and antibacterial agents. The synergistic action between relatively slow porin-mediated passive uptake across the outer membrane and active efflux transporters in the inner membrane creates a permeability barrier that reinforces the enzymatic modification barrier, which efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in small molecule translocation in Enterobacteriaceae and consider the crucial role of porins in antibiotic resistance. Commented [w1]: Is this specification necessary here?, in my opinion it deviates, better to put later… Commented [JP2]: Editor request... porins represent the preferred route for the entry of β-lactams, including cephalosporins, penicillins and carbapenems 14-16. The clinical relevance of membrane-associated mechanisms (MAMs) of resistance (i.e. porin defects and/or overexpression of multidrug efflux pumps) has been well established for these antibiotics. The Influx and Efflux rates control the internal concentration of antibiotics and represent the first lane (mechanical barrier) protecting the bacterial cells against therapeutic treatment 1-3,6. Consequently, studies on bacterial porins are receiving a renewed interest due to their key role in the bacterial susceptibility towards clinically used antibiotics. In combination with the expression of antibiotic-modifying enzymes expressed in the periplasm (e.g. β-lactamases), porins play a key role in β-lactam resistance 4,17. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in antibiotic translocation in Enterobacteriaceae. We explore structural aspects and the insights gained into permeation and the pore translocation process, the regulation of porin expression as well as the role of porins in the emergence of antibiotic susceptibility. Enterobacterial general porins Structural aspects The crystal structures of a general porin from Rhodobacter capsulatus 18 , the OmpF and PhoE porins from E. coli 19 and other E. coli OmpF structures including mutants 20,21 were the first to be solved. Only a limited number of other enterobacterial porin structures have been reported, i.e. E. coli OmpC, K. pneumoniae OmpK36 and Salmonella typhi OmpF 22-24. The lack of data has hindered attempts to relate structure to function. Recently, the structures of two porins from P. stuartii as well as the structures of the OmpF and OmpC orthologs of K. pneumoniae, E. aerogenes and E. cloacae have been reported 12,25,26. Another recent study reported th...