The importin ␣/␤ pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin ␣. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin ␣ and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin ␣, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin ␣-dependent NLSs provide useful information for identifying NLSs.

Background: Intramembranous cleavages of ␤-carboxyl-terminal fragment (␤CTF) by ␥-secretase generate amyloid ␤-protein (A␤). Results: Three-to six-residue peptides are released successively along with A␤ generation by lipid raft-associated ␥-secretase. Conclusion: ␥-Secretase cleaves ␤CTF through multiple interactive pathways for stepwise successive processing to generate A␤.Significance: This cleavage model provides insights into the precise molecular mechanism of A␤ generation.

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.

Extensive neuronal loss and aggregation of tau as cytoplasmic inclusions in neurons and glial cells in selected cortical and subcortical regions is the most striking characteristic of frontotemporal dementia and parkinsonism linked to chromosome 17 , which is caused by exonic or intronic mutations in the tau gene. Here , we examined the effects of four exonic mutations in four-repeat tau using stably transfected Chinese hamster ovary cells. The proportion of polymerized tubulin was the largest in the P301L transfectant. G272V and P301L transfectants showed greater instability of microtubules in the presence of Colcemid than wild-type tau , V337M , or R406W transfectants. Thus no distinct phenotypes were shared by the mutant tau transfectants with regard to microtubule assembly and stability. Unexpectedly , R406W showed low and negligible levels of phosphorylation at Thr 231 and Ser 396, respectively , in the transfectant. This presents a sharp contrast to the observation that tau aggregates in R406W-affected brains are heavily phosphorylated at these two sites. This result suggests that hyperphosphorylation at these sites cannot occur in the tau R406W bound to microtubules , and thus that the hyperphosphorylated species of tau may be generated only after disruption of microtubules. 1,2 Further studies using the candidate gene approach demonstrated six missense mutations and four intronic mutations in the tau gene in FTDP-17 families. 3-7These findings clearly indicate that these tau mutations are necessary and sufficient for the tau aggregation and neuronal loss observed in FTDP-17.Tau, a microtubule (MT)-associated protein (MAP), binds to tubulin at three or four repeats of 31 or 32 amino acids each, called the MT-binding domain, located in the carboxyl half of tau. In adult human brain, six isoforms containing three or four repeats are generated by alternative mRNA splicing from a single gene. Six known missense mutations in tau in FTDP-17 are located within or close to the MT-binding domain, and the intronic mutations are in the 5Ј splice site of exon 10, and thought to be involved in the formation of a stem loop, resulting in an increased proportion of four-repeat tau. 4 -6 Regarding exonic mutations, it is tempting to speculate that these mutations in tau interfere with its interaction with tubulin and thus decrease its ability to promote tubulin assembly.3,4 Along this line of investigation, two groups recently reported independently that the exonic mutations cause significant defects in MT assembly in the cell-free system. 8,9 However, acute depletion of tau was reported to have no effects on MT dynamics in cultured neurons 10 and, further, tau-knockout mice showed remarkably subtle effects on neurogenesis and neuronal organization. 11These results suggest that the tau mutation effects on MT assembly found in the cell-free system may not always be problematic within the cells or in vivo. Accordingly, it is too early to speculate about the pathogenesis of FTDP-17 based on the results from the cell-free sy...

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes.