N‐Tail translocation in a eukaryotic polytopic membrane protein

We have used the natural N-glycosylation site in the N-tail of cig30, a eukaryotic polytopic membrane protein, as a marker for N-tail translocation across the microsomal membrane. Analysis of C-terminally truncated cig30 constructs reveals that the first transmembrane segment is sufficient for translocation of the wild-type N-tail; in contrast, in a mutant with four arginines introduced into the N-tail the second transmembrane segment is also required for efficient N-tail translocation. Our observations imply a non-sequential assembly mechanism in which the ultimate location of the N-tail relative to the membrane may depend on more than one transmembrane segment.