Abstract
Importin α1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as "major" and "minor." The major site is located between ARM repeats 2-4, whereas the minor site spans ARM 7-8. In this study, we have characterized the cellular localization of human phospholipid scramblase 4 (hPLSCR4), a member of the phospholipid scramblase protein family. We identified a minimal NLS in hPLSCR4 ((273)GSIIRKWN(280)) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg(277), correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin α1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin α. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin α1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.
Highlights
Other members of the Phospholipid scramblases (PLSCR) protein family contribute to the intracellular calcium-triggered transbilayer movement of phospholipids that is observed in activated platelets and a variety of injured or apoptotic cells [2,3,4,5,6]
Sequence alignment of human PLSCR isoforms reveals that three motifs important for hPLSCR1 function are conserved in human phospholipid scramblase 4 (hPLSCR4) (Fig. 1)
Unlike the hPLSCR1-nuclear localization signals (NLSs), this putative NLS lacks a basic residue at the predicted P2 position (Lys258 in hPLSCR1-NLS) [11], which in all classical NLSs is essential for binding to importin ␣ and nuclear import [18]
Summary
Biochemical Techniques—Human PLSCR4 cDNA was cloned into the pZEOSV2(ϩ/Ϫ) plasmid using restriction sites NcoI and HindIII. To generate non-diffusible import cargos, we cloned the DNA region encoding hPLSCR4-NLS (residues 271–283) or hPLSCR1-NLS (residues 255–268) between restriction sites NcoI and BamHI of an engineered pGEX-4T-GFP vector (GE Healthcare) that encodes the green fluorescent protein (GFP) [30]. Both plasmids were expressed in Escherichia coli BL21(DE3) cells at 18 °C. Further cycles of positional and isotropic B-factor refinement with two TLS domains (for ⌬IBB-importin ␣ and the hPLSCR4-NLS, respectively) resulted in a final Rwork/Rfree ϳ18.7/22.6% (the Rfree was calculated using 5% of the observed reflections) (Table 1). Coordinates for the ⌬IBB-importin ␣1⁄7hPLSCR4-NLS complex were deposited in the Protein Data Bank with accession code 3Q5U
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