Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.
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 alpha2), importin beta, 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.
Gascard
P
P
Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. PDGascard@lbl.gov
Nunomura
W
W
Lee
G
G
Walensky
L D
LD
Krauss
S W
SW
Takakuwa
Y
Y
Chasis
J A
JA
Mohandas
N
N
Conboy
J G
JG
D016475:3T3 Cells / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D019556:COS Cells / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005091:Exons; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009479:Neuropeptides; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D020033:Protein Isoforms; D011770:Pyruvate Kinase / Q000378:metabolism; D013347:Subcellular Fractions; D014162:Transfection; D028901:alpha Karyopherins
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D028901:alpha Karyopherins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; C087978:karyopherin alpha 2; D011770:Pyruvate Kinase