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An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.
Journal article

An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.

  • Ungricht R Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
  • Pawar S Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
  • Kutay U Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland. ulrike.kutay@bc.biol.ethz.ch.
  • 2016-05-06
Published in:
  • Methods in molecular biology (Clifton, N.J.). - 2016
In vitro reconstitution
Inner nuclear membrane
Membrane protein
Microscopy
Nuclear envelope
Semi-permeabilized cell system
Cell Line
Gene Expression
Gene Order
Genes, Reporter
HeLa Cells
Humans
Membrane Proteins
Microscopy, Fluorescence
Nuclear Envelope
Recombinant Fusion Proteins
Time-Lapse Imaging
English Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/153590
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