Abstract
The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61alpha, Sec61beta and Sec61gamma, 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. Sec61alpha 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. Sec61alpha is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61alpha membrane-integration intermediates, we find: (i) newly synthesized Sec61alpha is adjacent to known components of the ER membrane-insertion site, namely Sec61alpha, Sec61beta and TRAM, and thus the integration of Sec61alpha appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61alpha remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61beta) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61alpha requires ER targeting by the signal-recognition particle.