Competition between neighboring topogenic signals during membrane protein insertion into the ER

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N‐terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild‐type protein, the N‐terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N‐terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N‐tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N‐terminal tail causes TM1 to adopt an orientation with its N‐terminal end in the cytoplasm. We suggest that the topology of the TM1‐TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology.