Stop-and-Move of a Marginally Hydrophobic Segment Translocating across the Endoplasmic Reticulum Membrane

Many membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane via the protein-conducting channel, the so-called translocon. The hydrophobic transmembrane segment of the translocating nascent polypeptide chain stops at the translocon and then moves laterally into the membrane. Partitioning of the hydrophobic segment into the membrane is the primary determinant for membrane insertion. Here, we examined the behavior of a marginally hydrophobic segment at the translocon and found that its stop-translocation was greatly affected by the C-terminally attached ribosomes. The marginally hydrophobic segment first stops at the membrane and then moves into the lumen as long as the nascent chain is attached to translating ribosomes. When it is released from the ribosome by the termination codon, the marginally hydrophobic segment does not move. Puromycin or RNase treatment also suppressed movement. The movement was reversibly inhibited by high-salt conditions and irreversibly inhibited by ethylenediaminetetraacetic acid. There is an unstable state prior to the stable membrane insertion of the transmembrane segment. This characteristic state is maintained by the synthesizing ribosome. [Display omitted] •Hydrophobic segment of membrane protein is integrated into membrane via translocon.•A marginally hydrophobic segment first stops at translocon and then moves into lumen.•The movement is observed only when the chain is attached to translating ribosome.•It is reversibly inhibited by high salt and irreversibly inhibited by ethylenediaminetetraacetic acid.•The membrane insertion is not decided rapidly even after stop-translocation.