Importance of transmembrane segments in Escherichia coli SecY

To assess the functional importance of the transmembrane regions of SecY, we constructed a series of SecY variants, in which the six central residues of each transmembrane segment were replaced by amino acid residues from either transmembrane segment 3 or 4 of LacY. The SecY function, as assessed by the ability to complement cold-sensitive secYmutants with respect to their growth and translocase defects, was eliminated by the alterations in transmembrane segments 2, 3, 4, 7, 9 and 10. Among them, those in segments 3 and 4 had especially severe effects. In contrast, transmembrane segments 1, 5, 6, and 8 were more tolerant to the sequence alterations. The purified protein with an altered transmembrane segment 6 retained, in large measure, the ability to support SecA-dependent preprotein translocation in vitro. These results will help us to further understand how the SecYEG protein translocation channel functions.