Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α

Abstract

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the ␤-karyopherin importin ␤, which mediates interactions with the nuclear pore complex, and the adaptor protein importin ␣, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.In eukaryotic cells, the genetic material and transcriptional machinery of the nucleus are separated from the translational machinery and metabolic systems of the cytoplasm by the nuclear envelope. This segregation facilitates the precise regulation of cellular processes such as gene expression (1), signal transduction (2), and cell cycle progression (3) through selective regulation of bidirectional transport between the nucleus and the cytoplasm. However, this physical separation also necessitates the existence of molecular machinery that specifically recognizes cargo in one compartment, translocates it through the nuclear pore, and releases it in the other compartment. Nuclear transport systems of this kind were first proposed when a nuclear targeting signal in the simian virus 40 (SV40) 2 large T antigen was characterized more than 20 years ago (4, 5). Since then, several pathways for nucleocytoplasmic transport have been described, of which the classical nuclear import pathway is the best characterized. The integration of detailed structural information on the components of the pathway, whole genome surveys, and extensive molecular analysis has generated powerful insight into the crucial interactions that underlie this pathway. Here we review recent studies that have defined key aspects of the cargo/import receptor interaction in the classical nuclear import cycle and present results of a bioinformatics-based assessment of the likely prevalence of this system within the model eukaryotic organism, Saccharomyces cerevisiae. Overview of Nucleocytoplasmic TransportTransport of macromolecules into and out of the nucleus occurs through large, proteinaceous structures called nuclear pore complexes (NPCs) (6 -9). Nuclear pore complexes allow passive diffusion of ions and small proteins (Ͻ40 kDa) but restrict passage of larger molecules to those containing an appropriate targeting signal (10, 11). The pores are constructed from a class of proteins called "nucleoporins," a subset of which contains a tandem series of phenylalanine-glycine (FG) repeats that line the central transport channel of the pore (8,(12)(13)(14).The active transport of macromolecular cargo between the cytoplasmic and nuclear compartments is facilitated by specific soluble carrier proteins. Th...

Identification of Siah-interacting protein as a potential regulator of apoptosis and curcumin resistance

“…We further demonstrate that phosphorylation at the Ser141 residue, the N-terminal region (aa 1-43) and the putative NLS region (aa 143-159) are crucial for nuclear translocation of SIP. These data are consistent with the contention that the nuclear import of most signaling proteins is NLS-dependent and is regulated by protein modifications, such as phosphorylation (Jans and Hubner, 1996;Lange et al, 2007). Interestingly, although curcumin-resistant subline MOLT-4/AraG shows defect in both SIP phosphorylation and nuclear translocation, it contains an SIP-WT sequence identical to that in MOLT-4, according to our direct sequencing data (data not shown).…”

“…As we know, small proteins (MW o40 kDa) may, in principle, diffuse passively through the nuclear pores (Lange et al, 2007). However, it has been shown that SIP can self-dimerize through the N-terminal aa 1-47 (Santelli et al, 2005).…”

Active nuclear import and cytoplasmic retention of activation-induced deaminase

“…The N-terminal domain contributes at least three of the four non-contiguous, positively charged determinants that we propose conform the AID NLS. Binding to importin-α adaptors, which mediate the nuclear import of proteins with classical basic NLS 29,30,36 , is in line with this. Without the 3D structure of an AID-importin complex it is difficult to assign which residues make direct contact and which play a structural role in displaying those residues.…”

“…4 b). This kind of NLS is recognized by members of the importin-α family of adaptors 29,36 . Indeed, AID-Flag was pulled down from extracts of Ramos cells by GST-importins-α1, α3 and α5 (Fig.…”

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“…Classical NLSs (cNLS) can bind to importin α through either a major site, a minor site, or both 8, 9. Monopartite cNLSs consist of a single cluster of positively charged residues, primarily lysines or arginines, that assume an ordered state once bound to importin α 10…”