Identification of a karyopherin β1/β2 proline-tyrosine nuclear localization signal in huntingtin protein

Abstract

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

FIGURE 1.

Huntingtin amino acids 174–207 contain elements of a karyopherin β2 basic PY NLS. The defined consensus for karyopherin β2-type NLSs is shown in A, alongside sequence alignments of the huntingtin NLS displaying full conservation of the basic rich KEIKK region, arginine 200, and proline-tyrosine PY 206/207 (boxed in gray) in B. The 18-amino acid intervening sequence flanked by proline turns, highlighted by a boxed outline, is also conserved. Below, the PY-NLS of ribonuclease P/MRP POP1 subunit is shown, with highly similar A and B sites. C, huntingtin 174–207 was cloned in-frame with GFP and β-galactosidase, a >120-kDa reporter for live cell expression. Sample images show huntingtin NLS as a GFP-β-galactosidase fusion, compared with no NLS, M9 hnRNPA1 NLS, and the KEIKK sequence. D, quantification of data in C, n = ∼50 cells quantified per construct. *, p < 0.01. Error bars, S.E.

FIGURE 2.

Huntingtin PY-NLS basic region is critical for NLS activity in vivo. A, positive control of GFP-Htt NLS-β-galactosidase, compared with P207A/Y208A and K177A/K178A mutants, quantified in B. Ctrl is GFP-β-galactosidase, which has been quantified and then set as base line to 0. C and D, analysis of huntingtin NLS mutants in the (Q17)1–465-eYFP context. n = ∼50 cells quantified per construct. *, p < 0.01. Error bars, S.E.

FIGURE 3.

Huntingtin PY-NLS utilizes the karyopherin β2/β1 import pathways. A, comparison of huntingtin NLS-mediated nuclear entry in human HEK293 versus mouse STHdh cells, quantified in B. C, overexpression of mCherry-karyopherin β2(541–890) (ΔRan binding) restoring wild-type NLS activity in HEK 293 cells (f–h), but not K177A/K178A mutant NLS (j–l). D, quantification of 3C. E, affinity chromatography of STHdh cell lysates on huntingtin NLS peptide-coupled columns. Column elutions at 0.5 m NaCl were separated by SDS-PAGE and Western blotted for karyopherin β2 (anti-Kapβ2,D45). Lysate lane contains 1/20 of column input. Beads indicates the beads-alone column. F, comparison of the SV40 NLS, M9 NLS, and huntingtin NLS in response to overexpression of M9M peptide competitor to karyopherin β2. G, overexpression of karyopherin β1(256–876) (ΔRan binding) increasing nuclear entry via the huntingtin NLS, but not a K177A/K178A mutant NLS. All NLSs are in GFP-β-galactosidase context in STHdh cells unless otherwise stated. For all quantification, n = ∼50 cells per construct. *, p < 0.01. Error bars, S.E.

FIGURE 4.

Huntingtin NLS 18-amino acid intervening sequence has a β sheet structure and cytoplasmic targeting activity. A, CD spectroscopy of the huntingtin IVS peptide, NH₂-PRSLRAALWRFAELAHLVRP-COOH, showing 44% β sheet characteristics. B, effect of IVS mutants on huntingtin NLS activity in the context of eGFP-X-β-galactosidase. Note the single proline substitution A187P, ΔAA(187,188) deletion, and double leucine substitution L195A/L198A. C, comparison of nuclear localization of CFP alone (a), CFP-IVS (c) and CFP-IVS L195A/L198A (b) in STHdh cells. Leucines 195 and 198 are critical for targeting to cytoplasm. Area in panel c is magnified in panel d to highlight targeting to cytoplasmic structures. n = ∼70 cells per construct. #, p values that are statistically significant compared with the wild-type huntingtin NLS (p < 0.01). *, p values that are statistically significant compared with Ctrl (p < 0.01). N.S. is nonsignificant compared with control. Error bars, S.E.