In:
Molecular Biology of the Cell, American Society for Cell Biology (ASCB), Vol. 10, No. 6 ( 1999-06), p. 1783-1798
Abstract:
The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R 80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R 80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R 80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R 80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin α2), importin β, and GTPase Ran. Quantitative analysis of protein–protein interactions using a resonant mirror detection technique showed that 4.1R 80 bound to Rch1 in vitro with high affinity (K D = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R 80 .
Type of Medium:
Online Resource
ISSN:
1059-1524
,
1939-4586
DOI:
10.1091/mbc.10.6.1783
Language:
English
Publisher:
American Society for Cell Biology (ASCB)
Publication Date:
1999
detail.hit.zdb_id:
1474922-1
SSG:
12
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