Mapping the functional domains of the Golgi stacking factor GRASP65.
The Golgi reassembly stacking protein (GRASP) family has been implicated in the stacking of Golgi cisternae and the regulation of Golgi disassembly/reassembly during mitosis in mammalian cells. GRASP65 is a dimer that can directly link adjacent surfaces through trans-oligomerization in a mitotically regulated manner. Here we show that the N-terminal GRASP domain (amino acids 1-201) is both necessary and sufficient for dimerization and trans-oligomerization but is not mitotically regulated. The C-terminal serine/proline-rich domain (amino acids 202-446) cannot dimerize nor can it link adjacent surfaces. It does, however, confer mitotic regulation on the GRASP domain through multiple sites phosphorylated by the mitotic kinases, cdc2/B1, and the polo-like kinase. Transient expression corroborated these results by showing that the GRASP domain alone inhibited mitotic fragmentation of the Golgi apparatus.
Wang
Yanzhuang
Y
Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut 06520, USA. yanzhuang.wang@yale.edu
Satoh
Ayano
A
Warren
Graham
G
D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008938:Mitosis; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D000097869:Polo-Like Kinase 1
D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D005982:Glutathione Transferase; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins; D008565:Membrane Proteins; D011392:Proline; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012694:Serine; D000097869:Polo-Like Kinase 1; C108705:GORASP1 protein, human