The role of GRASP65 in Golgi cisternal stacking and cell cycle progression.
In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans-oligomers. These conclusions, however, require further confirmation in the cell. In this study, we showed that the first 112 amino acids at the N-terminus (including the first PDZ domain, PDZ1) of the protein are sufficient for oligomerization. Systematic electron microscopic analysis showed that the expression of non-regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. These results provided evidence and a molecular mechanism by which GRASP65 stacks Golgi cisternal membranes. Further experiments revealed that inhibition of mitotic Golgi disassembly by expressing non-regulatable GRASP65 mutants did not affect equal partitioning of the Golgi membranes into the daughter cells. However, it delayed mitotic entry and suppressed cell growth; this effect was diminished by dispersing the Golgi apparatus with Brefeldin A treatment prior to mitosis, suggesting that Golgi disassembly at the onset of mitosis plays a role in cell cycle progression.
Tang
Danming
D
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109-1048, USA.
Yuan
Hebao
H
Wang
Yanzhuang
Y
D020126:Brefeldin A / Q000494:pharmacology; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D019281:Dimerization; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D018797:Cell Cycle Proteins; C108705:GORASP1 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D020126:Brefeldin A