Proteins actively transported into the nucleus via the classical nuclear import pathway contain nuclear localization signals (NLSs), which are recognized by the family of importin ␣ molecules. Importin ␣ contains 10 armadillo (arm) repeats, of which the N-terminal arm repeats 2-4 have been considered as the "major" NLS binding site. Interferon-activated, dimerized signal transducers and activators of transcription (STAT1 and STAT2) directly bind to importin ␣5 via a dimeric nonclassical NLS. Here we show by site-directed mutagenesis that the very C-terminal arm repeats 8 and 9 of importin ␣5 form a unique binding site for STAT1 homodimers and STAT1-STAT2 heterodimers. Influenza A virus nucleoprotein also contains a nonclassical NLS that is recognized by the C-terminal NLS binding site of importin ␣5, comprising arm repeats 7-9. Binding of influenza A virus nucleoprotein to importin ␣3 also occurs via the C-terminal arm repeats. Simian virus 40 large T antigen instead binds to the major N-terminal arm repeats of importin ␣3, indicating that one importin ␣ molecule is able to use either its N-or C-terminal arm repeats for binding various NLS containing proteins.Regulated import of molecules into the nucleus through the nuclear pores is a vital event in eukaryotic cells. Importins (also known as karyopherins) are the major cargo carriers from the cytoplasm into the nucleus. Large molecules (Ͼ40 kDa) that cannot passively diffuse through the nuclear pores use a signal-mediated transport system. The importin ␣/importin ␤-mediated import pathway was the first one to be discovered, and it is also referred to as the classical pathway. Proteins transported into the nucleus contain nuclear location signals (NLSs) 1 that are recognized by importin ␣/importin ␤ heterodimers. Importin ␣ recognizes and binds the NLS, and importin ␤ docks the complex to the nuclear pore and translocates it into the nucleus (1-3).Six different human importin ␣ molecules have been identified: importin ␣1 (Rch1, hSRP1␣), importin ␣3 (Qip1), importin ␣4 (hSRP1␥), importin ␣5 (hSRP1, NPI-1), importin ␣6, and importin ␣7 (4 -9). Importin ␣ proteins have remained structurally and functionally conserved throughout evolution. The six human importin ␣ proteins show over 60% sequence similarity.The crystal structures of two importin ␣ molecules, yeast importin ␣ (10) and mouse importin ␣2 (11), have been determined. Importin ␣ is composed of a large central domain that consist of 10 tandemly repeated armadillo (arm) motifs, which are organized in a superhelix flanked by small N-and Cterminal domains. The 10 arm repeats generate an array of binding pockets that are situated within a long helical surface groove. One binding pocket typically includes a tryptophan residue, followed by an asparagine residue 4 residues downstream. The arm repeats are variable within one protein, but they are remarkably conserved in sequence and order when homologous proteins from yeast to humans are compared. The N-terminal importin ␤ binding domain of importin ␣ mediates bi...

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The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age-related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti-aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti-aging activities for several phytochemicals and open up opportunities for the development of novel anti-aging therapies.

The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-␣, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-␣ family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-␣3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase-green fluorescent protein-RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-␣ or importin-␤ or by the absence of importin-␣. Binding assays using recombinant importin-␣1, -␣3, -␣4, -␣5, and -␣7 revealed a preferential interaction of the RanBP3 NLS with importin-␣3 and -␣4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-␣3. These results demonstrate that members of the importin-␣ family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.