NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle‐dependent reformation of the ER network

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Abstract
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/α and γ‐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.