DETERMINATION OF THE TRANSMEMBRANE TOPOLOGY OF YEAST SEC61P, AN ESSENTIAL COMPONENT OF THE ENDOPLASMIC-RETICULUM TRANSLOCATION COMPLEX

Sec61p is a highly conserved integral membrane protein that plays a ro le in the formation of a protein-conducting channel required for the t ranslocation of polypeptides into, and across, the membrane of the end oplasmic reticulum. As a major step toward elucidating the structure o f the endoplasmic reticulum translocation apparatus, we have determine d the transmembrane topology of Sec61p using a combination of C-termin al reporter-domain fusions and the in situ digestion of specifically i nserted factor Xa protease cleavage sites. Our data indicate the prese nce of 10 transmembrane domains, including several with surprisingly l imited hydrophobicity. Furthermore, we provide evidence for complex in tramolecular interactions in which these weakly hydrophobic domains re quire C-terminal sequences for their correct topogenesis, The incorpor ation of sequences with limited hydrophobicity into the bilayer may pl ay a vital role in the formation of an aqueous membrane channel requir ed for the translocation of hydrophilic polypeptide chains.