Figure 7.

A temporal role for the central cytoplasmic loop in membrane insertion. The open translocon/ribosome complex allows cotranslational insertion of the N-terminal six helices of lac permease. In the wild type, during translation of the central cytoplasmic loop, the N-terminal six helices move laterally from the translocon into the bilayer (A). Subsequently, the C-terminal six helices are translocated across the bilayer and inserted into the membrane. In the Δ mutants, as the length of the hydrophilic loop is progressively shortened, efficient insertion and activity are decreased appropriately. Likewise, by artificially shortening the length of the hydrophilic segment between helices VI and VII by inserting hydrophobic stretches (sites I and II), insertion and activity are also reduced in a context-dependent fashion. In the Δ20 mutant (B), helix VII enters the translocon before clearance of the N-terminal six helices, thereby blocking further insertion and leading to degradation of the permease before insertion into the bilayer. This phenotype is reversed either by insertion of a stretch of random hydrophilic amino acid residues or by introducing a split (site III).