Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers

Patrick Walker,Detlef Doenecke,Joerg Kahle

doi:10.1074/jbc.m806820200

Abstract

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

Highlights

Classical nuclear localization signals (NLS) are imported by a heterodimer of importin ␣ and ␤ [3, 4]
The CHRAC-15/17 Complex Is Recognized and Imported by Importin 13—As part of the human chromatin accessibility complex CHRAC [17], the CHRAC-15/17 complex fulfills its function in the nuclear compartment. This and the close relationship to the histone fold-containing NF-YB/NF-YC heterodimer, which is imported into the nucleus by importin 13 [10], led to the question of whether the CHRAC15/17 complex or histone fold heterodimers in general are recognized by importin 13
The immobilized GST-CHRAC-15/His-CHRAC-17 complex was incubated with preassembled importin ␣/␤, importin ␤, importin 5, importin 7, importin 9, and importin 13

Summary

Introduction

Classical NLS are imported by a heterodimer of importin ␣ and ␤ [3, 4]. Proteins with a nonclassical NLS are directly recognized by one import receptor without the help of the importin ␣ adapter molecule. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

Results

Conclusion