NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.
The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.
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
Kaneko
Yayoi
Y
Uchiyama
Keiji
K
Hosokawa
Nobuko
N
Nagata
Kazuhiro
K
Murata
Masayuki
M
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D016466:CHO Cells; D002453:Cell Cycle / Q000502:physiology*; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism* / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006056:Golgi Apparatus / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism
D000906:Antibodies; D009687:Nuclear Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C470473:Vcpip1 protein, rat; D000251:Adenosine Triphosphatases; C404942:p97 ATPase