Ordered membrane insertion of an archaeal opsin in vivo

The prevailing model of polytopic membrane protein insertion is based largely on the in vitro analysis of polypeptide chains trapped during insertion by arresting translation, To test this model under conditions of active translation in vivo, we have used a kinetic assay to determine the order and timing with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon Halobacterium salinarum, BO is the apoprotein of bacteriorhodopsin, a structurally well characterized protein containing 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 domains of the protein. Cysteine translocation during BO translation was monitored by pulse-chase radiolabeling and rapid derivatization with a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The results show that the N-terminal domain, the BC loop, and the FC loop are translocated in order 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 gel shift, The translocation order was confirmed by applying the assay to mutant proteins containing two cysteines in separate extracellular domains, Comparison of the translocation results with in vivo measurements of BO elongation 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 archaeal polytopic membrane protein insertion in vivo.