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 role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.
Wilkinson
B M
BM
School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom.
Critchley
A J
AJ
Stirling
C J
CJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D015245:Deoxyribonuclease BamHI; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D026901:Membrane Transport Proteins; D008861:Microsomes / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015245:Deoxyribonuclease BamHI; D015951:Factor Xa