The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding͞bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMP␤. The SRY-R133W mutant shows reduced IMP␤ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMP␤ binding, whereas SRY-R75N and SRY-R76P show normal IMP␤ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Dengue virus NS5 protein is a multifunctional RNAdependent RNA polymerase that is essential for virus replication. We have shown previously that the 37-amino acid interdomain spacer sequence (residues 369 X 2 KKX 14 KKKX 11 RKX 3 405 ) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, ␤-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369 -389 or residues 386 -405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369 -389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin ␣/␤ heterodimeric nuclear import receptor. We also show that the importin ␤-binding site (residues 320 -368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin ␤, and able to target ␤-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin ␤ in the infectious cycle.Dengue virus is a member of the genus Flavivirus within the family Flaviviridae that also contains the genera Pestivirus and Hepacivirus. There are around 70 viruses grouped in the Flavivirus genus, which includes yellow fever virus (YFV), 1 Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, and tick-borne encephalitis virus. Dengue virus causes a benign syndrome known as Dengue fever and a more severe illness, Dengue hemorrhagic fever or in its severest form Dengue shock syndrome (1-3). There are four serologically and phylogenetically distinguishable Dengue viruses (types 1-4), and the disease they cause is of substantial world wide significance to human health but is mostly restricted to tropical and sub-tropical areas because of its transmission by the Aedes aegypti mosquito (4).Flaviviruses possess a single-strand, positive-sense RNA genome of around 11 kb, which is capped but not polyadenylylated, and encodes a single polyprotein including three structural and seven non-structural proteins in the order C-prM-E-NS1-NS2A-NS3-NS2B-NS2A-NS4B-NS5 (5, 6). Replication of flaviviruses occurs at membrane-associated replicase complexes localized in the perinuclear region. The replicase complex has been extensively characterized in several flaviviruses and includes NS1, NS2A, NS3, NS4A, and NS5 (7-12) and possibly some cellular proteins (13-15). Whereas the protein and RNA interactions of the replicase complexes still require detailed characterization, several recent studies (7, 17) have focused on the structure and function of the NS3 and NS5 proteins.NS3 (69 kDa) is a multifunctional protein that has been shown to have protease, helicase, NTPase, and 5Ј-terminal RNA triphosphatase activities (16 -19). NS5 (104 kDa) contains a well characterized RNA-dependent RNA polymerase ac...

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.