Ordered membrane insertion of an archaeal opsin in vivo

The prevailing model of polytopic membrane protein insertion is based large ly on the in vitro analysis of polypeptide chains trapped during insertion by arresting translation, To test this model under conditions of active tra nslation in vivo, we have used a kinetic assay to determine the order and t iming with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon Halobacterium salinarum, BO is the apopro tein of bacteriorhodopsin, a structurally well characterized protein contai ning seven transmembrane alpha-helices (A-G) with an N-out, C-in topology, H. salinarum strains were constructed that express mutant BO containing a C -terminal His-tag and a single cysteine in one of the four extracellular do mains of the protein. Cysteine translocation during BO translation was moni tored by pulse-chase radiolabeling and rapid derivatization with a membrane -impermeant, sulfhydryl-specific gel-shift reagent. The results show that t he N-terminal domain, the BC loop, and the FC loop are translocated in orde r from the N terminus to the C terminus, Translocation of the DE loop could not be examined because cysteine mutants in this region did not yield a ge l shift, The translocation order was confirmed by applying the assay to mut ant proteins containing two cysteines in separate extracellular domains, Co mparison of the translocation results with in vivo measurements of BO elong ation indicated that the N-terminal domain and the BC loop are translocated cotranslationally, whereas the FG loop is translocated posttranslationally . Together, these results support a sequential, cotranslational model of ar chaeal polytopic membrane protein insertion in vivo.