Nuclear import pathway of the telomere elongation suppressor TRF1: inhibition by importin alpha.
Telomere repeat factor 1 (TRF1) regulates the steady-state length of chromosomes, whereby its overexpression results in telomere shortening while dominant negative TRF1 mutations can lead to telomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleus at mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism by which TRF1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, in turn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism of TRF by expressing and purifying a recombinant TRF1-GFP (green fluorescent protein) fusion protein that is functional in terms of being able to bind telomeric DNA specifically as shown using a novel, quantitative double-label gel mobility shift assay. We quantitate the ability of TRF1-GFP to accumulate in the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathway of TRF1 is mediated by importin (Imp) beta1 and Ran. Imp beta is shown to bind directly to TRF1 with nanomolar affinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experiments also demonstrate that Imp beta residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerized to Imp beta, Imp alpha was found to inhibit Imp beta-mediated nuclear accumulation, although not affecting Imp beta binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1; that its nuclear import is mediated directly by Imp beta but inhibited by Imp alpha may represent a novel regulatory mechanism, with potential relevance to oncogenesis.
Forwood
Jade K
JK
Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra City, Australia.
Jans
David A
DA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D035321:Telomeric Repeat Binding Protein 1; D028901:alpha Karyopherins / Q000502:physiology*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D035321:Telomeric Repeat Binding Protein 1; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins