Stop-transfer efficiency of marginally hydrophobic segments depends on the length of the carboxy-terminal tail.

Hydrophobic stop-transfer sequences generally serve to halt the translocation of polypeptide chains across the endoplasmic reticulum membrane and become integrated as transmembrane alpha-helices. Using engineered glycosylation sites as topology reporters, we show that the length of the nascent chain between a hydrophobic segment and the carboxy terminus of the protein can affect stop-transfer efficiency. We also show that glycosylation sites located close to a protein's C terminus are modified in two distinct kinetic phases, one fast and one slow. Our findings suggest that membrane integration of a hydrophobic segment is not simply a question of thermodynamic equilibrium, but can be influenced by details of the translocation mechanism.