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DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex
1Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
The Journal of Biological Chemistry
|September 25, 1998
Summary
Loss of the DAD1 protein destabilizes the oligosaccharyltransferase (OST) complex, impacting N-linked glycosylation and protein stability in eukaryotic cells. This study reveals DAD1
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Modification
Background:
- Asparagine-linked glycosylation (N-linked glycosylation) is a crucial, conserved protein modification in eukaryotes.
- The oligosaccharyltransferase (OST) complex, located in the endoplasmic reticulum (ER), catalyzes N-linked glycosylation.
- The mammalian OST complex comprises ribophorin I, ribophorin II, OST48, and the recently identified DAD1 subunit.
Purpose of the Study:
- To investigate the functional role of the DAD1 subunit within the mammalian OST complex.
- To examine the consequences of DAD1 loss on OST subunit stability and N-linked glycosylation.
- To utilize a temperature-sensitive cell line (tsBN7) with a DAD1 mutation to study OST complex dynamics.
Main Methods:
- Western blot analysis of cell lysates from tsBN7 cells shifted to a non-permissive temperature.
- Monitoring of steady-state levels of OST subunits (RI, RII, OST48, DAD1) and other ER proteins (TRAP, Sec61).
- Assessment of N-glycosylation status of ribophorins and a secretory glycoprotein.
Main Results:
- Degradation of DAD1 at the non-permissive temperature led to reduced levels of OST48 and ribophorins.
- Other ER translocation components (TRAP, Sec61) remained unaffected, indicating OST-specific instability.
- N-glycosylation of ribophorins and a secretory glycoprotein was significantly impaired following DAD1 loss.
Conclusions:
- DAD1 is essential for the stability of the OST complex, including OST48 and ribophorins.
- Loss of DAD1 results in the functional inactivation of the OST complex and widespread N-glycosylation defects.
- The findings highlight DAD1's critical role in maintaining OST complex integrity and cellular glycosylation homeostasis.
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