The importin 13 binding platform is conserved between different histone fold

In addition to the CHRAC-15/17 complex, we also wanted to verify the role of the conserved positively charged amino acid residues for the nuclear import of the related histone fold pairs, p12/CHRAC-17 and NC2α/NC2β. The NC2α/NC2β (NC2) complex binds to the promoter-bound TATA-binding protein (TBP) and the formation of the NC2-TBP complex results in a sterical hindrance for the recruitment of the general transcription factors IIB and IIA (Goppelt et al., 1996; Kamada et al., 2001; Mermelstein et al., 1996). Hence, the NC2 complex functions as a transcriptional repressor (Inostroza et al., 1992; Meisterernst and Roeder, 1991). As recently

FIG. 21: Nuclear import of NC2α/NC2β is mediated by importin 13 and depends on basic amino acids.

(A) Conserved basic amino acids in cNLS deficient (∆cNLS) NC2α and NC2β were progressively substituted. The

numbers (1-4) represents the order of consecutive mutations against alanine (see also Fig. 19A and 19B).

(B) HeLa P4 cells were transiently transfected with plasmid DNA encoding flag-importin 13. Additionally, EGFP-NC2α and RFP-NC2β, both mutated in their cNLS by site-directed mutagenesis and then consecutively mutated in their basic amino acids as indicated, were cotransfected. The subcellular localization of the NC2α/NC2β

92 80

65 55

41

8 20

35 45

59

0 20 40 60 80 100

NC2α ∆cNLS NCSβ ∆cNLS

Mut 1 Mut 1

Mut 2 Mut 2

Mut 3 Mut 3

Mut 4 Mut 4

% cells

cyto.

nucl.

shown in our group (Kahle et al., 2009) both NC2 subunits, NC2α and NC2β, contain a cNLS.

The NC2 complex is therefore recognized and imported by the importin α/β heterodimer.

However, when the cNLSs in NC2α (⁴KKKK⁷) and NC2β (¹⁰⁰KRRRK¹⁰³) are mutated via a substitution of K5 in NC2α and R101 in NC2β against alanine (in Fig. 21 referred to as ∆cNLS), the NC2 complex is still imported into the nucleus in an importin 13-dependent pathway (Fig. 21B, upper row). To analyze the influence of the conserved basic amino acids in the NC2 complex (Fig. 21A), EGFP-NC2α and RFP-NC2β, both deficient of its cNLS and progressively mutated in their basic amino acids were coexpressed with importin 13 in HeLa P4 cells. The subcellular distribution was detected by direct fluorescence. For a quantitative analysis, the fluorescence intensity in cotransfected cells was measured and the ratio between cytoplasmic and nuclear localization was calculated (Fig. 21C). Similar to the changes observed in the subcellular localization of CHRAC-15/17 complex, the transfection of the NC2α/NC2β complex also led to a decreased nuclear uptake when progressively mutated at the respective sites (Fig. 21B). The strong nuclear accumulation of the cNLS deficient NC2 complex (92 %), was lost when three basic amino acids on each subunit were mutated leading to a homogenous localization (55 %).

When eight basic amino acids in the cNLS deficient NC2 complex were mutated, 59 % of the complex accumulated in the cytoplasm.

Finally we analyzed the nuclear transport of the p12/CHRAC-17 complex deficient in conserved basic amino acids. Analogous to previous transfection experiments with CHRAC-15/17 HeLa P4 cells were cotransfected with plasmid DNA coding for importin 13 and mutated p12 and CHRAC-17 (Fig. 22). Although the overall effect of progressive substitution of basic residues was less dramatic compared to mutated CHRAC-15/17 and NC2α/NC2β, nuclear transport of p12/CHRAC-17 was reduced strongly if four basic amino acids on each subunit were mutated (Fig. 22A and B). Additionally, we performed in vitro GST-Pulldown assays with mutated p12/CHRAC-17 complexes (Fig. 22C). Again, we found no evidence for reduced heterodimerization, caused by the substitution of R45 and R69 for alanine in p12. The loss of basic amino acids in the p12/CHRAC-17 complex resulted in decreased importin 13 binding,

FIG. 21: continued

DNA was counterstained with Hoechst. Colocalization of the red RFP-fusion protein and the green EGFP-fusion protein is shown in yellow (merge). The loss of conserved basic amino acids in cNLS deficient NC2α and NC2β results in an increased cytoplasmic accumulation of the NC2complex in transfected cells. (C) The subcellular distribution of colocalized cNLS-deficient (∆cNLS) and mutated NC2α/NC2β complexes was quantitatively analyzed using the program ImageJ (NIH). The fluorescence intensity of 20 cotransfected cells was measured and the ratio between nuclear (nucl.) and cytoplasmic (cyto.) localization was calculated. R, arginine; K, lysine;

FIG. 22: Basic amino acids conserved among the H2A/H2B histone fold family are necessary for the interaction between importin 13 and the p12/CHRAC-17 heterodimer. (A) HeLa P4 cells were transiently cotransfected with plasmid DNA encoding wild type (wt) or stepwise mutated EGFP-p12 and RFP-CHRAC-17 as indicated. Flag-importin 13 was additionally coexpressed. Subcellular localization of the p12/CHRAC-17 heterodimer was detected 24 hours post transfection by direct fluorescence. Colocalization of the fluorescently-labelled subunits is shown in yellow (merge). DNA was stained with Hoechst. The loss of basic amino acid residues in p12 and CHRAC-17 does change the subcellular distribution of the heterodimer towards a homogeneous localization. (B) The subcellular distribution of colocalized wild type (wt) and mutated p12/CHRAC-17 complex was quantitatively analyzed using the program ImageJ (NIH). The fluorescence intensity of 20 cotransfected cells

was measured and the ratio between nuclear (nucl.) and cytoplasmic (cyto.) localization was calculated.

(C) Recombinant GST-p12/His-CHRAC-17 wild type heterodimer (wt) and heterodimers with alanine substitutions of conserved basic amino acids were incubated with recombinant, purified importin 13. Binding was determined in the absence (-) or presence (+) of RanGTP (Q69L-mutant). 20 % of the applied importin 13 is shown as input.

Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. Substitution of basic residues in p12 and CHRAC-17 led to a decreased binding of importin 13 to the p12/CHRAC-17 complex. wt, wild type;

R, arginine; K, lysine; A, alanine; Mw, molecular weight; imp13, importin 13.

88 87 81 72

similar to the observations made for the mutated CHRAC-15/17 complex (Fig. 22C and see again Fig. 20C). In more detail, upon the substitution of three conserved basic amino acids against alanine in each histone fold subunit, p12 and CHRAC-17, binding of importin 13 was lost completely.

3.13 The importin 13 binding platform in histone fold pairs is created by