title: Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane.
abstract: Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention-based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo.
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland Molecular Life Sciences PhD Program, CH-8057 Zurich, Switzerland.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Ungricht
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Klann
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Horvath
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland ulrike.kutay@bc.biol.ethz.ch.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kutay
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D000097682:Lamin B Receptor
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097682:Lamin B Receptor; C413025:SUN2 protein, human; C093413:lamina-associated polypeptide 2