Nuclear Import Pathway of the Telomere Elongation Supressor TRF1: Inhibition by Importin
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- 作者：Jade K. Forwood and David A. Jans
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 30, 2002
- 年：2002
- 卷：41
- 期：30
- 页码：9333 - 9340
- 全文大小：223K
- 年卷期：v.41,no.30(July 30, 2002)
- ISSN：1520-4995
文摘
Telomere repeat factor 1 (TRF1) regulates the steady-state length of chromosomes, wherebyits overexpression results in telomere shortening while dominant negative TRF1 mutations can lead totelomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleusat mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism bywhich TRF1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, inturn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism ofTRF by expressing and purifying a recombinant TRF1-GFP (green fluorescent protein) fusion proteinthat 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 accumulatein the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathwayof TRF1 is mediated by importin (Imp) 
2.gif" BORDER=0 ALIGN="middle">1 and Ran. Imp2.gif" BORDER=0 ALIGN="middle"> is shown to bind directly to TRF1 with nanomolaraffinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experimentsalso demonstrate that Imp2.gif" BORDER=0 ALIGN="middle"> residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerizedto Imp2.gif" BORDER=0 ALIGN="middle">, Imp
was found to inhibit Imp2.gif" BORDER=0 ALIGN="middle">-mediated nuclear accumulation, although not affecting Imp2.gif" BORDER=0 ALIGN="middle">binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1;that its nuclear import is mediated directly by Imp2.gif" BORDER=0 ALIGN="middle"> but inhibited by Imp may represent a novel regulatorymechanism, with potential relevance to oncogenesis.
2.gif" BORDER=0 ALIGN="middle">1 and Ran. Imp2.gif" BORDER=0 ALIGN="middle"> is shown to bind directly to TRF1 with nanomolaraffinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experimentsalso demonstrate that Imp2.gif" BORDER=0 ALIGN="middle"> residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerizedto Imp2.gif" BORDER=0 ALIGN="middle">, Imp was found to inhibit Imp2.gif" BORDER=0 ALIGN="middle">-mediated nuclear accumulation, although not affecting Imp2.gif" BORDER=0 ALIGN="middle">binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1;that its nuclear import is mediated directly by Imp2.gif" BORDER=0 ALIGN="middle"> but inhibited by Imp may represent a novel regulatorymechanism, with potential relevance to oncogenesis.