Negatively charged residues in the IgM stop-transfer effector sequence regulate transmembrane polypeptide integration

A non-hydrophobic sequence that contributes to the biogenesis of a transmem brane protein is termed a stop-transfer effector (STE), To examine the mech anism of STE-mediated stop-transfer, a series of fusion proteins were const ructed containing variants of a putative STE: from murine IgM fused to an o therwise translocated hydrophobic sequence. Unexpectedly, the fraction of m olecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a r eporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonst rating that the STE sequence can be located at either side of the endoplasm ic reticulum membrane. Our results suggest that recognition of a broad stru ctural feature formed primarily by negatively charged residues within the S TE halts translocation and triggers membrane integration, even when the neg ative charges end up on the cytoplasmic side of the membrane. Since functio nal STE sequences photocross-link to two membrane proteins not previously i dentified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function.