Abstract
We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biological Transport, Active
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CDC2 Protein Kinase / metabolism*
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CHO Cells
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Cell Line, Tumor
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Cricetinae
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Cytosol / metabolism
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Endoplasmic Reticulum / metabolism*
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Golgi Apparatus / metabolism*
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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Leukemia L5178 / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mice
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Mitosis
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Phosphorylation
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
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Vesicular Transport Proteins / genetics
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Vesicular Transport Proteins / metabolism
Substances
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Membrane Proteins
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Recombinant Fusion Proteins
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Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
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Vesicular Transport Proteins
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Green Fluorescent Proteins
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CDC2 Protein Kinase