Membrane integration of Sec61α:: a core component of the endoplasmic reticulum translocation complex

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61 alpha, Sec61 beta and Sec61 gamma, at least two of which (alpha and beta) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61 alpha functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61 alpha is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a crosslinking approach to analyse the environment of a series of discrete Sec61 alpha membrane-integration intermediates, we find: (i) newly synthesized Sec61 alpha is adjacent to known components of the ER membrane-insertion site, namely Sec61 alpha, Sec61 beta and TRAM, and thus the integration of Sec61 alpha appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61 alpha remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61 beta) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61 alpha requires ER targeting by the signal-recognition particle.