Role of CRM1 in signal-mediated nuclear protein export
- 作者：Ossareh-Nazari ; Batool ; Bachelerie ; Franç ; oise ; Dargemont ; Catherine
- 刊名：Biology of the Cell
- 出版年：1998
- 期刊代码：11_02484900
- 类别：imm
- 出版时间：January, 1998
- 卷：90
- 期：1
- 页码：113
- 文件大小：141 K
摘要
Leucine-rich amino acid sequences responsible for efficient protein nuclear export (NES) have recently been identified in an increasing number of proteins, in particular, human immunodeficiency virus- type 1 Rev protein, the protein kinase A, and the inhibitor IkappaBα (IκBα). To analyze the molecular mechanisms govering NES-dependent nuclear protein export, we developed an assay that reconstitutes nuclear export in vitro. Hela cells were transiently transfected with cDNAs encoding fusion proteins consisting of Myc-tagged pyruvate kinase, wild-type or mutated IκBα NES, and SV40 large T antigen NLS (NLS-PK-NES and NLS-PK-NES mut, respectively). Cells were then treated with digitonin to permeabilize the plasma membrane without affecting the integrity of the nuclear envelope. Export of NLS-PK-NES and NLS-PK-NESmut were analyzed under different incubation conditions by indirect immunofluorescence and western blot. In this assay nuclear export was found to be NES, extracts and energy dependent. The replacement of total extracts by the recombinant proteins required for nuclear import (karyopherins, Ran/TC4, and p10) promoted the nuclear import of a karyophilic substrate, but did not induce the nuclear export of NLS-PK-NES, indicating that an essential component for nuclear export was provided by the total extracts. To identify this component, we used a drug, leptomycin B, shown to inhibit HIV Rev export in vivo. Leptomycin B was able to block NES-dependent protein export in the vitro assay, suggesting that this drug inhibits a component involved in nuclear export. In Schizosaccharomyces pombe, the target of leptomycin B is the CRM1 protein whose human homolog has recently been identified. CRM1 is a nuclear protein that shares sequence homology with karyopherin b familly. We cloned human CRM1 and found that in vitro translated CRM1 was able to bind specifically NES sequence. The CRM1-NES interaction was inhibited by leptomycin B in dose dependent manner. These data indicate that CRM1 acts as a NES receptor in protein nuclear export. Like the karyopherin b familly, CRM1 is able to bind Ran-GTP in a cooperative way with NES. By analogy with the nuclear import process, Ran or a Ran-binding protein may also regulate the interaction of CRM1-NES-containing protein complexes with the nuclear pore complex.