ABSTRACT
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.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/chemistry , Protein Structure, Secondary , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Deoxyribonuclease BamHI , Factor Xa/metabolism , Fungal Proteins/chemistry , Intracellular Membranes/metabolism , Intracellular Membranes/ultrastructure , Membrane Proteins/biosynthesis , Membrane Transport Proteins , Microsomes/metabolism , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Plasmids , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Restriction Mapping , SEC Translocation Channels , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae ProteinsABSTRACT
Searches of the protein data bases revealed limited homologies between several regions of the human erythrocyte glucose transporter containing a relative abundance of hydrogen-bonding amino-acid side chains, and proteins of the NADH-ubiquinone oxidoreductase family. This raised the possibility the binding sites for glucose and ubiquinone may be similar in the respective proteins. Experimental studies demonstrated that ubiquinone Q0 does in fact inhibit both glucose entry and glucose exit in human erythrocytes with kinetics consistent with the existence of ubiquinone binding sites at both the exofacial and endofacial sides of the transporter. Glucose transport was also inhibited by the water-soluble tryptophan-inactivating agent, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide, and this is consistent with the presence of tryptophan residues in two of the exofacial amino-acid sequences proposed as candidates for involvement in glucose binding sites.
