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-α. 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.

Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3

“…8D), which makes a distance of 1-2 aa for the spatial distance between the two basic clusters that interact with the minor binding site and major binding site in importin-a, respectively. A previous report also showed that Ty1 integrase contains a bona fide bipartite NLS with a 29-aa linker (37). Our data, together with these reports, reveal that the traditional definition of NLS is too restrictive; the linker length can vary depending on amino acid composition and conformation.…”

Cofilin Nuclear-Cytoplasmic Shuttling Affects Cofilin-Actin Rod Formation During Stress

“…The intervening sequence between the two basic rich sequences in a bipartite NLS is generally 10-12 amino acids long. In the cofilin NLS, this intervening sequence is shorter than predicted, however, it has been reported that the number of amino acids between the basic regions of a bipartite NLS can vary greatly, being either longer (Lange et al, 2010) or shorter (Taniguchi et al, 2002) than the earlier defined consensus. When we mutated both basic regions of the bipartite NLS (AASSTPEEVKAAKK) the nuclear localization signal was not functional, either out of context or in context of full length cofilin.…”

Crystal Structure of Rice Importin-α and Structural Basis of Its Interaction with Plant-Specific Nuclear Localization Signals

“…The solid-phase binding assay was performed essentially as described previously (Matsuura et al, 2003;Lange et al, 2010;Takeda et al, 2011;Marfori et al, 2012). The assay was performed on Immuno MaxiSorp 96-well plate (Thermo Fisher Scientific).…”