A subunit of the mammalian oligosaccharyltransferase, DAD1, interacts with Mcl-1, one of the bcl-2 protein family.
DAD1 is a mammalian homologue of Saccharomyces cerevisiae Ost2p, a subunit of the oligosaccharyltransferase complex. Loss of its function induces apoptosis in hamster BHK21 cells. By means of a two-hybrid method involving DAD1 as bait, the C-terminal region of Mcl-1, one of the bcl-2 family, was isolated. Consistently, DAD1 binds well to Mcl-1 in COS cells when overexpressed. On deletion analysis, the C-terminal domain of Mcl-1 containing BH(2) (bcl-2 homologous domain) was found to be essential for the interaction with DAD1. On the other hand, the C-terminal half of DAD1 was concluded to be essential for the interaction with Mcl-1. Surprisingly, a DeltaC-DAD1 mutant lacking only 4 amino acid residues from the C-terminus did not complement the tsBN7 mutation, while it interacted well with Mcl-1. In contrast, DeltaN-DAD1 lacking 20 amino acid residues from the N-terminus still exhibited the ability to complement the tsBN7 mutation. Thus, the C-terminus of DAD1 was suggested to play an important role in N-linked glycosylation and to complement the tsBN7 mutation. Mcl-1 may be required for the inhibition of apoptotic cell death caused by a loss of DAD1.
Makishima
T
T
Department of Molecular Biology, Research Institute for Diseases of the Chest, Graduate School of Medical Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Yoshimi
M
M
Komiyama
S
S
Hara
N
N
Nishimoto
T
T
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D002478:Cells, Cultured / Q000166:cytology; D017931:DNA Primers / Q000737:chemistry; D005822:Genetic Vectors; D006031:Glycosylation; D006602:Hexosyltransferases; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D021122:Protein Subunits; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014166:Transferases / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C083331:DAD1 protein, human; D017931:DNA Primers; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D021122:Protein Subunits; D019253:Proto-Oncogene Proteins c-bcl-2; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D001616:beta-Galactosidase