The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport.
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.
Cushman
Ian
I
Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Stenoien
David
D
Moore
Mary Shannon
MS
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D000546:Amanitins; D000903:Antibiotics, Antineoplastic; D018797:Cell Cycle Proteins; D002843:Chromatin; D005231:Fatty Acids, Unsaturated; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D003609:Dactinomycin; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein; C038753:leptomycin B