Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyl-transferase complex in Saccharomyces cerevisiae

被引:43
作者
Kim, H
Yan, Q
von Heijne, G
Caputo, GA
Lennarz, WJ [1 ]
机构
[1] SUNY Stony Brook, Dept Biochem & Cell Biol, Stony Brook, NY 11794 USA
[2] SUNY Stony Brook, Inst Cell & Dev Biol, Stony Brook, NY 11794 USA
[3] Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden
关键词
D O I
10.1073/pnas.1332735100
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Ost4p is a minimembrane protein containing only 36 amino acids and is a subunit of oligosaccharyltransferase (OT) in Saccharomyces cerevisiae. It was found previously when amino acid residues 18-25 of Ost4p were mutated to ionizable amino acids and defects were observed in the interaction between Ost4p and either Stt3p or Ost3p, two other components of OT. The transmembrane segment of Ost4p is likely to extend from residues 10-25. This is consistent with the finding that alpha-helicity is estimated to be 36% by CD analysis of synthetic Ost4p in liposomes. This value is in reasonable agreement with the assumption that amino acids 10-25 (16 of 36 or 44%) are transmembrane. Therefore, the mutation-sensitive region (residues 18-25) is localized to only one half of the putative transmembrane domain of Ost4p. To learn where this region of Ost4p is situated in relation to the faces of endoplasmic reticulum (ER) membrane, we determined the membrane topology of Ost4p using an in vivo method and established that it is an N-lumen-C-cyto, type I membrane protein. These results indicate that the mutation-sensitive region of Ost4p is localized in the cytoplasmic leaflet of the ER membrane. In the current study, we also observed a loss of direct interaction between Ost3p and Stt3p in the presence of ost4 temperature-sensitive mutants, which indicates Ost4p, via interactions with amino acid residues in the cytosolic leaflet of the ER membrane, functions to bind these two proteins together in a subcomplex of OT.
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页码:7460 / 7464
页数:5
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