The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase.
The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.
Kano
Fumi
F
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan.
Kondo
Hisao
H
Yamamoto
Akitsugu
A
Tanaka
Arowu R
AR
Hosokawa
Nobuko
N
Nagata
Kazuhiro
K
Murata
Masayuki
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005033:Ethylmaleimide / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology; D046529:Microscopy, Electron, Transmission; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D013301:Streptolysins / Q000494:pharmacology
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015843:Serpins; D013301:Streptolysins; C025964:streptolysin O; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D016203:CDC2 Protein Kinase; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; D005033:Ethylmaleimide; D015739:Nocodazole