Construction of Defined Polytopic Integral Transmembrane Proteins

Signal and stop transfer sequences are discrete regions within a polypeptide chain able to initiate or terminate translocation of the protein across the membrane of the endoplasmic reticulum. We have investigated the role of these topogenic sequences in the biogenesis of polytopic transmembrane proteins. Plasmids encoding various patterns of well-characterized signal and stop transfer sequences fused to a set of topogenically inert passenger domains were constructed. These molecules were expressed by transcription-translation in a cell-free system or by microinjection of transcripts into Xenopus oocytes. The observed orientation with respect to the membrane was dependent on the order of signal and stop transfer sequences in the coding region. These results were used to test the hypothesis that a protein can achieve polytopic transmembrane orientation using combinations of simple topogenic sequences. We conclude that some (but not all) patterns of signal and stop transfer sequences confer polytopic orientation to proteins across the membrane of the endoplasmic reticulum.