Journal article
Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R
Molecular biology of the cell, Vol.10(6), pp.1783-1798
06/1999
DOI: 10.1091/mbc.10.6.1783
PMCID: PMC25371
PMID: 10359596
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 4.1R80 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. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 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 4.1R80 bound to Rch1 in vitro with high affinity (KD = 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 4.1R80.
Details
- Title: Subtitle
- Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R
- Creators
- Philippe Gascard - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720Wataru Nunomura - Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, Tokyo 162, Japan; andGloria Lee - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720Loren D Walensky - The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205Sharon Wald Krauss - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720Yuichi Takakuwa - Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, Tokyo 162, Japan; andJoel A Chasis - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720Narla Mohandas - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720John G Conboy - Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720
- Contributors
- Peter Walter (Editor)
- Resource Type
- Journal article
- Publication Details
- Molecular biology of the cell, Vol.10(6), pp.1783-1798
- DOI
- 10.1091/mbc.10.6.1783
- PMID
- 10359596
- PMCID
- PMC25371
- NLM abbreviation
- Mol Biol Cell
- ISSN
- 1059-1524
- eISSN
- 1939-4586
- Publisher
- American Society for Cell Biology
- Language
- English
- Date published
- 06/1999
- Academic Unit
- Iowa Neuroscience Institute; Immunology; Internal Medicine
- Record Identifier
- 9984070469302771
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