Mechanism of Sec61-mediated insertion of proteins into the endoplasmic reticulum membrane

In eukaryotic cells, hydrophobic signal sequences of newly synthesized secretory and membrane proteins target them to the Sec61 translocon in the endoplasmic reticulum (ER) membrane. The translocon forms a hydrophilic pore, in its idle state closed by a lumenal plug domain and a hydrophobic constriction ring. Within the Sec61 channel, transmembrane segments of proteins achieve their proper orientation (topology) and are laterally released into the lipid bilayer. Orientation of signal sequences in the ER membrane is determined by charged residues flanking the hydrophobic core of the signal, hydrophobicity of the signal, the size and folding properties of the N-terminal domain preceding the signal and, in some cases, the length of the C-terminus.
In Part I of this thesis we compared the insertion process of N-terminal versus internal signal-anchors of single-spanning membrane proteins and determined the effect of the N-terminal hydrophilic domain on protein topogenesis. We showed that insertion of these two types of signals occurs via different mechanisms. Transition from N-terminal to internal signals, achieved by extension of the N-domain with hydrophilic residues, was accompanied by loss of C-terminal length dependence and insensitivity to increased hydrophobicity of the signal. It indicated that, in contrast to N-terminal signals, signal-anchors localized internally cannot undergo reorientation within the pore. Furthermore, hydrophilic N-terminal domains sterically hinder N-translocation.
In Part II we analyzed the insertion process of proteins with conflicting signal sequences: type I cleavable hemagglutinin (HA) signal and a type II signal-anchor of H1. We showed that proteins with wild-type HA and H1 signals, connected by a 40-amino acid linker compete for the preferred orientation in the translocon, manifested by a rapid inversion of a fraction of the polypeptides, triggered by the signal-anchor. The process could be slowed down by increasing the hydrophobicity of the H1 signal or manipulating its flanking charges. Under such conditions, topogenesis was interrupted upon termination of translation, like previously observed for N-terminal signal-anchors. In contrast to single-spanning membrane proteins, the topogenesis window is not a constant of the translocation machinery, but rather appears to be substrate-specific.
In Part III we tested the function of the apolar core of the Sec61 translocon. We mutated the ring residues of yeast Sec61p to more hydrophilic, bulky, or even charged amino acids (alanines, glycines, serines, tryptophans, lysines, or aspartates). The translocon turned out to be surprisingly tolerant even to the charge mutations in the constriction ring, since growth and translocation efficiency were not drastically affected. Ring mutants altered the integration of hydrophobic sequences into the lipid bilayer, which indicated that the translocon does not simply catalyze the partitioning of potential transmembrane segments between an aqueous environment and the lipid bilayer, but that it plays an active role in setting the hydrophobicity threshold for membrane integration.