title: An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore.
abstract: We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.
authors.affiliation: La Jolla Cancer Research Foundation, California 92037.
authors.forename: D D
authors.initials: DD
authors.lastname: Newmeyer
authors.forename: D J
authors.initials: DJ
authors.lastname: Forbes
mesh_terms: D000818:Animals; D002474:Cell-Free System; D003600:Cytosol / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology*; D005260:Female; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014981:Xenopus
chemical_list: D009687:Nuclear Proteins; D005033:Ethylmaleimide