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Vol. 10, Issue 6, 1783-1798, June 1999

Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

Philippe Gascard,^* Wataru Nunomura, Gloria Lee,^* Loren D. Walensky,^§¶ Sharon Wald Krauss,^* Yuichi Takakuwa, Joel A. Chasis,^* Narla Mohandas,^* and John G. Conboy^*

 ^*Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720;  Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo 162, Japan; and  ^§The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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 2), importin , 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 (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 4.1R⁸⁰.

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   Corresponding author. E-mail address: PDGascard{at}lbl.gov.
^¶   Present address: Children's Hospital, Boston, MA 02115.

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Molecular Biology of the Cell
Vol. 10, 1783-1798, June 1999
Copyright © 1999 by The American Society for Cell Biology

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