MEMBRANE INTEGRATION OF SEC61-ALPHA - 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 b eta) are adjacent to nascent proteins during membrane insertion. Anoth er component of the translocation machinery is the translocating chain -associating membrane (TRAM) protein, which is also adjacent to many n ascent proteins during membrane insertion. Sec61 alpha functions as th e major component of a transmembrane channel formed by oligomers of th e Sec61 complex. This channel is the site of secretory protein translo cation and membrane protein integration at the ER membrane. Sec61 alph a is a polytopic integral membrane protein, and we have studied its bi osynthesis and membrane integration in vitro. Using a crosslinking app roach to analyse the environment of a series of discrete Sec61 alpha m embrane-integration intermediates, we find: (i) newly synthesized Sec6 1 alpha is adjacent to known components of the ER membrane-insertion s ite, 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 transm embrane domain of Sec61 alpha remains adjacent to protein components o f 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.