title: An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.
abstract: 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.
authors.affiliation: Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland.|Molecular Life Sciences Ph.D. Program, CH-8057, Zurich, Switzerland.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Ungricht
authors.affiliation: Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland.|Molecular Life Sciences Ph.D. Program, CH-8057, Zurich, Switzerland.
authors.forename: Sumit
authors.initials: S
authors.lastname: Pawar
authors.affiliation: Department of Biology, Institute of Biochemistry, ETH Zurich, CH-8093, Zurich, Switzerland. ulrike.kutay@bc.biol.ethz.ch.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kutay
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D059008:Time-Lapse Imaging
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins
keywords: In vitro reconstitution; Inner nuclear membrane; Membrane protein; Microscopy; Nuclear envelope; Semi-permeabilized cell system