The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin ␣ through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS (ϳ3 kcal/mol) as well as the energy of inhibition of the importin ␣ importin ␤-binding domain (ϳ3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin ␣-importin ␤ complex. On this scale, a functional NLS has a binding constant of ϳ10 nM, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 M. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.The sequestering of genetic material in the nucleus by eukaryotic cells provides a powerful mechanism for the regulation of gene expression and other cellular processes through the selective translocation of proteins between the nucleus and the cytoplasm (1-3). Recently, the regulated transport of proteins across the nuclear envelope has been recognized as a crucial step in an increasing number of cellular processes (4 -6). Understanding the mechanisms of regulated protein translocation through nuclear pores requires a detailed definition of the signals that mark a macromolecular complex for nuclear import or export.The best characterized mechanism for translocation across the nuclear envelope is protein import which depends on the "classical" nuclear localization signal (NLS) 1 (7). This NLS consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10 -12 residues (bipartite) (8,9). This signal is recognized by the heterodimeric import receptor complex comprising importin ␣ and importin ␤ (3). Importin ␣ is an adapter protein that consists of a small N-terminal importin ␤-binding (IBB) domain and a larger Cterminal NLS-binding domain (10 -14). Importin ␤ does not directly interact with the NLS cargo but acts to direct importin ␣ to the nuclear pore (15, 16). In the absence of importin ␤, "NLS-like" sequences of the N-terminal IBB domain form an intramolecular bond with the NLS-binding site inhibiting the interaction between importin ␣ and the NLS cargo. Evidence for this auto-inhibition is found in the crystal structure of full-length importin ␣ as well as in vitro binding assays (16 -19). Thus, the interaction between importin ␣ and the NLS cargo is regulated by importin ␤. In an analogous m...

Nup98 is a component of the nuclear pore that plays its primary role in the export of RNAs. Nup98 is expressed in two forms, derived from alternate mRNA splicing. Both forms are processed into two peptides through autoproteolysis mediated by the C-terminal domain of hNup98. The three-dimensional structure of the C-terminal domain reveals a novel protein fold, and thus a new class of autocatalytic proteases. The structure further reveals that the suggested nucleoporin RNA binding motif is unlikely to bind to RNA. The C terminus also contains sequences that target hNup98 to the nuclear pore complex. Noncovalent interactions between the C-terminal domain and the cleaved peptide tail are visible and suggest a model for cleavage-dependent targeting of hNup98 to the nuclear pore.

Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin ␣, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin ␣, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.The segregation of the nuclear genetic material from the cytoplasmic machinery that translates it into proteins provides the eukaryotic cell with intricate mechanisms for controlling gene expression. This segregation, however, also presents the cell with a mechanistic problem. Because most intra-and extracellular signaling pathways culminate with changes in gene expression within the nucleus, signals must cross the nuclear envelope to gain access to the genetic material. This signal is almost invariably a protein, such as a transcription factor, that enters the nucleus. In addition, once a gene is transcribed, the messenger RNA must then be exported across the nuclear envelope into the cytoplasm where it is translated into protein.In fact, the nuclear envelope is a critical information barrier across which both RNA and proteins are selectively transported in a highly regulated manner to establish orderly communication and behavior within the cell (1).The best characterized mechanism for translocation across the nuclear envelope is protein import, which depends on the "classical" nuclear localization signal or NLS 2 (2). A classical NLS consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10 -12 residues (bipartite) (3, 4). NLS-containing cargoes are imported by a heterodimeric import receptor complex composed of importin ␣ and importin ␤ (5). Importin ␣, which recognizes and binds to the NLS sequence, is an adapter protein (6) that consists of a small N-terminal importin ␤-binding domain (IBB) and a larger C-terminal NLS-binding domain (7-11). Importin ␤ does not directly interact with the NLS cargo but instead targets importin ␣ to the nuclear pore (12, 13). In the absence of importin ␤, "NLS-like" sequences within the N-terminal IBB domain of importin ␣ form an intra-molecular bond with the NLS-binding site, which inhibits the interaction between importin ␣ and the NLS cargo (13)(14)(15...

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/ karyopherin ␣ and ␤. The importin ␣ subunit recognizes classical NLS sequences, and the importin ␤ subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin ␤ binding (IBB) domain of importin ␣ regulates NLS-cargo binding in the absence of importin ␤ in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin ␣ protein. These mutants dissect the two functions of the N-terminal IBB domain, importin ␤ binding and autoinhibition. One of these importin ␣ mutations, A3, decreases auto-inhibitory function without impacting binding to importin ␤ or the importin ␣ export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin ␣ remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin ␣ is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin ␣ to the cytoplasm.In eukaryotes, the nuclear envelope provides an essential barrier that separates the nuclear genome from the intermediary metabolism, signaling systems, and translation machinery of the cytoplasm. Selective bi-directional transport of macromolecules across this nuclear envelope regulates critical cellular processes such as gene expression (1, 2). All nucleocytoplasmic transport of macromolecules occurs through large proteinaceous structures, called nuclear pore complexes (NPC), 1 that perforate the nuclear envelope (3, 4). These macromolecular cargoes are specifically targeted to and transported through NPCs by a family of soluble nuclear transport receptors (5, 6).The small GTPase, Ran, governs the interactions between the nuclear transport receptors and macromolecular cargoes (5, 7). Import receptors bind cargo in the absence of RanGTP, whereas export receptors bind cargo in a trimeric complex with RanGTP (5,8). This mode of regulation requires an asymmetric distribution of RanGTP, with more RanGTP in the nucleus than in the cytoplasm. To achieve this asymmetry, the Ran regulatory proteins are compartmentalized with the GTPase activating protein (RanGAP), which generates RanGDP, in the cytoplasm (9) and the guanine nucleotide exchange factor (RCC1), which generates RanGTP, in the nucleus (10).The best-characterized nuclear import process occurs via receptor recognition of a classical nuclear localization signal (NLS). This classical NLS is typified by a cluster of basic amino acids (monopartite) or two clusters of basic amino acids separated by a 10 -12 amino acid linker (bipartite) (11, 12). A heterodimeric import receptor, composed of importins ␣ and ␤ (also known as karyopherin ␣ and ␤), mediates the nuclear import of proteins that contain a classical NLS (13-15). Over the last several years many...

We have developed a quantitative in vitro steady-state fluorescence depolarization assay to measure the interaction of a nuclear localization signal (NLS) substrate with its receptors. This assay relies on the change in fluorescence depolarization of an NLS fused to the green fluorescent protein upon binding to receptor. No binding is observed in the absence of a functional NLS, and binding affinities measured correlate with previous in vivo studies of NLS function. We have used this assay to test an auto-inhibitory model for the interaction of an NLS with the NLS receptor complex. This model suggests that NLS binding to importin ␣ is modulated by an auto-inhibitory sequence within the N terminus of importin ␣, which is displaced by importin ␤ binding. Consistent with this model, NLS substrates bind tightly to an N-terminally truncated importin ␣ lacking the autoinhibitory domain (K d ϳ10 nM), but measurable binding to full-length importin ␣ is only observed upon addition of importin ␤. Our quantitative results support the autoinhibitory model and suggest a mechanism for a switch between a cytoplasmic, high affinity and a nuclear, low affinity NLS receptor. This predicted mode of interaction would facilitate binding of substrate in the cytoplasm and its subsequent release into the nucleus.In eukaryotic cells, selective transport of proteins into the nucleus is mediated by short amino acid sequences that are referred to as nuclear localization signals (NLSs). 1 The "classical" NLS motif contains one (monopartite) or two (bipartite) clusters of basic amino acids (1). The monopartite NLS is exemplified by the NLS of SV40 large T-antigen ( 126 PKKKRKV 132 ), whereas the bipartite, consisting of two small clusters of basic residues separated by a linker sequence, is found in the NLS of nucleoplasmin ( 155 KRPAATKKAGQAKKKK 170 ) (2, 3).A heterodimeric receptor for the classical nuclear protein import pathway has been identified. This receptor consists of two proteins referred to as importin ␣ and importin ␤ (4). The molecular and biochemical characterization of importin ␣ shows that it recognizes and binds directly to the NLS peptide (5-8). Importin ␣ acts as an adapter in the formation of a trimeric import complex containing the NLS-bearing cargo, importin ␣, and the import receptor importin ␤. Importin ␤ interacts with components of the nuclear pore complex known as nucleoporins (9 -11). As a result of these interactions the import complex is targeted to the nuclear pore complex and then translocated into the nucleus (9, 12) via a process that requires the activity of the small GTPase Ran (4, 13).The mechanism for the recognition of NLS peptides by importin ␣ has been enigmatic because of the diversity in the amino sequences of experimentally defined NLSs. The structural basis for recognition of at least one type of NLS by Saccharomyces cerevisiae importin ␣ is now known from a crystallographic analysis of the NLS binding domain of the 50-kDa yeast importin ␣ fragment bound to an SV40 NLS peptide (8). This 50-kDa im...