514 publications from this institution
We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.
Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine is the translocon, which works in concert with ribosomes to manage the orderly insertion of alpha-helical membrane proteins directly into the endoplasmic reticulum membrane of eukaryotes or into the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid membrane, where physicochemical interactions determine the final three-dimensional structure. Much progress has been made during the past several years toward understanding the physical chemistry of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider the connection between the physical principles of membrane protein stability and translocon selection of transmembrane helices.
