A specific segment of the transmembrane domain of Wbp1p is essential for its incorporation into the oligosaccharyl transferase complex.
Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex.
Li
Guangtao
G
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Yan
Qi
Q
Oen
Handy O
HO
Lennarz
William J
WJ
D000595:Amino Acid Sequence; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006602:Hexosyltransferases; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000378:metabolism; D013696:Temperature; D014166:Transferases / Q000737:chemistry*
D008565:Membrane Proteins; D009844:Oligosaccharides; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase