Expanding the definition of the classical bipartite nuclear localization signal

Abstract

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-alpha. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.

Figure 1. Both basic regions, BR1 and BR2, are required for proper nuclear localization and retrotransposition of Ty1 integrase

A) Tools for investigating the requirements for nuclear localization of Ty1 IN NLS. The last 55 amino acids of Ty1 IN containing both BR1 and BR2 were fused to two tandem GFP molecules as shown. Using site-directed mutagenesis, alanine residues were substituted for the KKR sequence of BR1 (IN NLS_br1BR2), BR2 (IN NLS_BR1br2), or in combination (IN NLS_br1br2). B) Localization of GFP₂-IN NLS variants. Wildtype cells expressing GFP₂-IN NLS variants were analyzed by direct fluorescence microscopy. Hoechst dye was used to visualize the location of the nucleus. Corresponding DIC images are shown. C) As assessed using a previously described retrotransposition assay (56), retrotranposition is significantly decreased in Ty1 NLS br1BR2 and Ty1 NLS BR1br2 mutants. Recombination deficient cells (Δrad52) were transformed with the pAR100 test plasmid, which contains the either the wildtype Ty1 retrotransposon or Ty1 NLS variants (Figure 1A). The number of retrotransposition events obtained for wildtype integrase was set to 100%. Results were quantitated as described in Materials and Methods.

Figure 2. Localization of bipartite SV40 T3 NLS reporters containing various linker lengths

A) Wildtype cells expressing bpSV40 T3 NLS reporters containing artificial linkers of various lengths (number of amino acids in linker) were analyzed by direct fluorescence microscopy. Corresponding DIC images are shown. B) Importin-α mutant srp1-54 cells expressing bpSV40 T3 NLS reporters containing various linker lengths were analyzed by direct fluorescence microscopy. Nab2-GFP was localized as a control that is imported into the nucleus through a mechanism that does not depend on importin-α (35, 57). Corresponding DIC images are shown. C) Wildtype cells expressing bpSV40 T3 NLS reporters containing serine/glycine linkers of various lengths (number of amino acids in linker) were analyzed by direct fluorescence microscopy. Corresponding DIC images are shown. D) Wildtype cells expressing bpSV40 T3 NLS reporters containing alanine linkers of various lengths (number of amino acids in linker) were analyzed by direct fluorescence microscopy. Corresponding DIC images are shown.

Figure 3. The 29 amino acid linker from integrase is a functional bipartite linker sequence

A) Wildtype cells expressing GFP₂ (control), bpSV40 T3 NLS with the conventional 10 aa linker (10), a long 30 aa linker (30), the 29 aa integrase linker (IN), or a variant of the integrase linker (mutIN) were analyzed using direct fluorescence microscopy. Corresponding DIC images are shown. B) Importin-α mutant srp1-54 cells expressing bpSV40 T3 NLS (10), bpSV40 T3 NLS (IN), or control Nab2-GFP were analyzed using direct fluorescence microscopy. Corresponding DIC images are shown.

Figure 4. The 29 amino acid linker of integrase mediates binding to importin-α through a bipartite mechanism

Binding of purified ΔIBB-importin-α to bpSV40 T3 NLS cargoes was assessed using a quantitative in vitro binding assay (39). Purified GFP, bpSV40 T3 NLS-GFP (10), (30) or (IN) proteins were incubated with increasing concentrations of ΔIBB-importin-α and the change in absorbance (Δ Anisotropy) of the samples at 450 nm was measured. Using Prism5 software (GraphPad Software, Inc.), we generated non-linear regression binding curves to yield the K_D values provided in Table 2.

Figure 5. Analysis of putative bipartite cNLSs within candidate cellular proteins

A) Wildtype cells expressing integrated Csl4-GFP, Rrp4-GFP, or Mft1-GFP were analyzed by direct fluorescence microscopy. Corresponding DIC images are shown. B) Wildtype or srp1-54 cells expressing Csl4 NLS-GFP₂, GFP₂-Rrp4 NLS, Mft1 NLS-GFP₂, bpSV40 T3 NLS-GFP₂ (10), or Nab2-GFP were analyzed using direct fluorescence microscopy. Corresponding DIC images are shown.

Figure 6. The bipartite cNLS within Rrp4 is necessary for nuclear import

A) Wildtype cells expressing bpSV40 T3 NLS (10), bpSV40 T3 NLS (30), bpSV40 T3 NLS (IN), or bpSV40 T3 NLS reporters containing the putative long bipartite cNLS linkers from Rrp4 or Mft1 were analyzed using direct fluorescence microscopy. Corresponding DIC images are shown. B) Importin-α mutant srp1-54 cells expressing bpSV40 T3 NLS (10), bpSV40 T3 NLS (Rrp4), or Nab2-GFP (control) were analyzed using direct fluorescence microscopy. Corresponding DIC images are shown. C) Wildtype Rrp4 or Rrp4 containing amino acid substitutions within the putative bipartite cNLS (K122A/R123A, NLSmut1 or R149A/R150A/K151A, NLSmut2) was localized as a GFP-fusion protein in wildtype cells using direct fluorescence microscopy. Corresponding DIC images are shown.