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...

Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localization of integrase is dependent upon a C-terminal nuclear targeting sequence. However, the nuclear import machinery that recognizes this nuclear targeting signal has not been defined. We investigated the mechanism by which Ty1 integrase gains access to nuclear DNA as a model for how other retroelements, including retroviruses like HIV, may utilize cellular nuclear transport machinery to import their essential nuclear proteins. We show that Ty1 retrotransposition is significantly impaired in yeast mutants that alter the classical nuclear protein import pathway, including the Ran-GTPase, and the dimeric import receptor, importin-α/β. Although Ty1 proteins are made and processed in these mutant cells, our studies reveal that an integrase reporter is not properly targeted to the nucleus in cells carrying mutations in the classical nuclear import machinery. Furthermore, we demonstrate that integrase coimmunoprecipitates with the importin-α transport receptor and directly binds to importin-α. Taken together, these data suggest Ty1 integrase can employ the classical nuclear protein transport machinery to enter the nucleus.

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-a/Srp1, to the G 2 /M transition of the cell cycle. Here, we utilize two engineered mutants of importin-a/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G 1 to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G 1 /S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G 2 /M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-a mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G 1 /S cell cycle transition.