Dimerization of sterol regulatory element-binding protein 2 via the helix-loop-helix-leucine zipper domain is a prerequisite for its nuclear localization mediated by importin beta.

The sterol regulatory element-binding protein 2 (SREBP-2), a transcription factor of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family, is synthesized in the form of a membrane-attached precursor molecule. When cells are deprived of sterols, a two-step proteolytic processing releases the transcriptionally active N-terminal segment of SREBP-2, thereby allowing it to enter the nucleus. In previous studies, we showed that the nuclear import of SREBP-2 occurs via the direct interaction of importin beta with the HLH-Zip domain. In this study, in order to more completely understand the intracellular dynamics of SREBP-2, we focused on the manner by which importin beta recognizes SREBP-2 at the initial step of the import. It was found that the active form of SREBP-2 exists as a stable dimer in solution and that the substitution of leucine residues for alanine in the leucine zipper motif disrupted the dimerization. It was also demonstrated that this mutant protein did not enter the nucleus either in vivo or in vitro. Solution binding assays, which involved the chemical cross-linking of wild-type or mutated SREBP-2 with importin beta, revealed that the import-active complex appeared to be composed of a dimeric form of SREBP-2 and importin beta. In addition, the SREBP-2 binding domain of importin beta corresponded to an overlapping but not identical region for importin alpha binding, which may explain how importin beta is able to recognize the dimeric HLH-Zip directly. These results indicate that dimerization is a prerequisite process for the nuclear import of SREBP-2 mediated by importin beta.

Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip.

The sterol regulatory element-binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix-leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin beta. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin beta in the absence of importin alpha. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2-importin beta complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin beta in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix-leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin beta.

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Nuclear targeting of DNA.

The nuclear membrane is a tight barrier for cytoplasmic proteins, but nuclear proteins have the intrinsic ability to overcome this barrier by an active signal-mediated process. Specific cytoplasmic carrier proteins have the responsibility to escort these proteins into the nucleus through the nuclear pore. The nuclear membrane is also a tight barrier for exogenous DNA delivered by synthetic vehicles, while many of the karyophilic viruses have a mechanism to actively deliver their genome through the nuclear pore. Virus DNA and RNA cannot move into the nucleus by themselves and require the viral structural proteins for efficient nuclear transport. In this article, we review the recent progress in understanding the mechanism of the nuclear transport of proteins and the virus genome, and discuss the possibility of developing synthetic gene-delivery systems based on these outcomes.

Transcriptional feedback and definition of the circadian pacemaker in Drosophila and animals.

The modern era of Drosophila circadian rhythms began with the landmark Benzer and Konopka paper and its definition of the period gene. The recombinant DNA revolution then led to the cloning and sequencing of this gene. This work did not result in a coherent view of circadian rhythm biochemistry, but experiments eventually gave rise to a transcription-centric view of circadian rhythm generation. Although these circadian transcription-translation feedback loops are still important, their contribution to core timekeeping is under challenge. Indeed, kinases and posttranslational regulation may be more important, based in part on recent in vitro work from cyanobacteria. In addition, kinase mutants or suspected kinase substrate mutants have unusually large period effects in Drosophila. This chapter discusses our recent experiments, which indicate that circadian transcription does indeed contribute to period determination in this system. We propose that cyanobacteria and animal clocks reflect two independent origins of circadian rhythms.

Nucleocytoplasmic protein transport and recycling of Ran.

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.

Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal.

A monoclonal antibody, 5H4, that recognizes human herpesvirus 7 (HHV-7) was used in Western analysis to probe HHV-7-infected SupT1 cells. This antibody recognizes a 40-kDa virus-specific polypeptide that is expressed in the absence of viral DNA synthesis. By screening a lambdagt11 HHV-7 cDNA library, the gene encoding the protein was identified as the U27 open reading frame previously reported [J. Virol. (1996) 70, 5975-5989]. Immunofluorescent studies showed a punctate nuclear localization of the protein in both HHV-7-infected cells and transfected cells. A computer program predicted two classic nuclear localization signals (NLSs) in the middle and C-terminal regions of the protein. A C-terminal deletion mutant of the protein could not enter the nucleus, whereas green fluorescent protein or maltose binding protein fused to the C-terminal region of the protein was transported into the nucleus. These findings demonstrate that the predicted C-terminal, but not middle, NLS of the protein actually function as NLS. In addition, nuclear transport of a maltose binding protein-fusion protein containing the C-terminal NLS of the U27 protein was inhibited by both wheat germ agglutinin and a Q69L Ran-GTP mutant, indicating that the U27 protein is transported into the nucleus from the cytoplasm by means of classic nuclear transport machinery. Interestingly, this NLS motif is highly conserved at the C-termini of all herpesvirus DNA polymerase processivity factors that have been examined.

Asymmetric crossing over in the spontaneous formation of large deletions in the tonB-trp region of the Escherichia coli K-12 chromosome.

The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA+ and recA- cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79 x 10(-9) and 1.09 x 10(-9) for recA+ and recA- cells, respectively. We analyzed 12 deletions from recA+ and 10 from recA- cells by cloning and direct sequencing. The deletions ranged in size from 5612 bp to 15142 bp for recA+ and from 5428 bp to 13289 for recA- cells. Three deletions from recA+ cells and five deletions from recA- cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the repeat in the mutant sequence. Seven deletions from recA+ cells and three deletions from recA- cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini in the wild-type are characterized by short (2-4 bp) repeats. From these results, a model is presented for the generation of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp.