The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase

• Published in: Genes to cells : devoted to molecular & cellular mechanisms Vol. 10; no. 4; pp. 333 - 344
• Main Authors: Kano, Fumi, Kondo, Hisao, Yamamoto, Akitsugu, Tanaka, Arowu R., Hosokawa, Nobuko, Nagata, Kazuhiro, Murata, Masayuki
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.04.2005
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Animals; Bacterial Proteins - pharmacology; CDC2 Protein Kinase - metabolism; CHO Cells; Cricetinae; Cricetulus; Cytosol - metabolism; Cytosol - ultrastructure; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Ethylmaleimide - pharmacology; Green Fluorescent Proteins - genetics; Guanosine Triphosphate - metabolism; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Membrane Fusion - physiology; Microscopy, Electron, Transmission; Mitosis; Nocodazole - pharmacology; Nuclear Proteins - metabolism; Permeability; Phosphorylation; Recombinant Fusion Proteins - metabolism; Serpins - genetics; Serpins - metabolism; Streptolysins - pharmacology
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1111/j.1365-2443.2005.00837.x

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.