Studies on the role of the hydrophobic domain of Ost4p in interactions with other subunits of yeast oligosaccharyl transferase.
In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits. Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex. To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues. This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37 degrees C and OT activity measured in vitro were impaired. In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT. These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane.
Kim
H
H
Department of Biochemistry, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Park
H
H
Montalvo
L
L
Lennarz
W J
WJ
D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005656:Fungal Proteins / Q000378:metabolism*; D006602:Hexosyltransferases; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase