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Mapping the functional domains of the Golgi stacking factor GRASP65
Yanzhuang Wang1, Ayano Satoh, Graham Warren
1Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut 06520, USA. yanzhuang.wang@yale.edu
The Journal of Biological Chemistry
|December 4, 2004
Summary
The Golgi reassembly stacking protein (GRASP) N-terminal domain enables dimerization and linking, while the C-terminal domain confers mitotic regulation through phosphorylation, impacting Golgi structure during cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The Golgi reassembly stacking protein (GRASP) family is crucial for Golgi cisternae stacking and cell division.
- GRASP65 facilitates Golgi structure maintenance through dimerization and trans-oligomerization.
Purpose of the Study:
- To delineate the functional domains of GRASP65 responsible for dimerization, trans-oligomerization, and mitotic regulation.
- To investigate the role of specific protein domains in Golgi dynamics during mitosis.
Main Methods:
- Protein domain analysis of GRASP65.
- Biochemical assays for dimerization and trans-oligomerization.
- Phosphorylation site mapping using mitotic kinases (cdc2/B1, polo-like kinase).
- Transient expression studies in mammalian cells.
Main Results:
- The N-terminal GRASP domain (1-201) is necessary and sufficient for dimerization and trans-oligomerization, independent of mitotic regulation.
- The C-terminal domain (202-446) lacks dimerization capacity but confers mitotic regulation via phosphorylation.
- Mitotic kinases cdc2/B1 and polo-like kinase phosphorylate the C-terminal domain, regulating GRASP65 function.
- Expression of the GRASP domain alone inhibited Golgi fragmentation during mitosis.
Conclusions:
- GRASP65 function is regulated by distinct N-terminal and C-terminal domains.
- Mitotic regulation of GRASP65 involves phosphorylation of its C-terminal domain by specific kinases.
- Understanding GRASP65 domain function provides insights into Golgi apparatus dynamics during cell division.