The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport

Mol Biol Cell. 2004 Jan;15(1):245-55. doi: 10.1091/mbc.e03-06-0409. Epub 2003 Oct 17.

Abstract

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or alpha-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Active Transport, Cell Nucleus / physiology*
  • Amanitins / pharmacology
  • Animals
  • Antibiotics, Antineoplastic / pharmacology
  • Cell Cycle Proteins*
  • Cells, Cultured
  • Chromatin / metabolism*
  • Cricetinae
  • Cytosol / metabolism
  • Dactinomycin / pharmacology
  • Fatty Acids, Unsaturated / pharmacology
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Guanosine Triphosphate / metabolism
  • Microinjections
  • Microscopy, Fluorescence
  • Nuclear Proteins*
  • Protein Synthesis Inhibitors / pharmacology
  • Recombinant Fusion Proteins / metabolism
  • Transcription, Genetic / physiology
  • ran GTP-Binding Protein / metabolism*

Substances

  • Amanitins
  • Antibiotics, Antineoplastic
  • Cell Cycle Proteins
  • Chromatin
  • Fatty Acids, Unsaturated
  • Guanine Nucleotide Exchange Factors
  • Nuclear Proteins
  • Protein Synthesis Inhibitors
  • Recombinant Fusion Proteins
  • Dactinomycin
  • Guanosine Triphosphate
  • ran GTP-Binding Protein
  • leptomycin B