Identification of specific binding proteins for a nuclear location sequence

Adam, Stephen A.; Lobl, Thomas J.; Mitchell, Mark A.; Gerace, Larry

doi:10.1038/337276a0

Cited by 217 publications

(154 citation statements)

References 22 publications

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“…Consistent with this finding, nuclear p34cdc2 kinases were activated, in vitro, depending on an addition of the purified E. coli produced cdc25C protein. These findings, together with the previous finding that a normal traffic through the nuclear pore is essential for the activation of p34cdc2 kinase by tyrosine dephosphorylation (Adam et al, 1989;Shi and Thomas, 1992;Stochaj and Silver, 1992). A cell-cycle specific nuclear transfer of protein has been reported for cyclin B in HeLa cells , which was transferred into the nuclei in G2 phase.…”

Section: Discussionsupporting

confidence: 76%

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Seki

Yamashita

Nishitani

et al. 1992

MBoC

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We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein. The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function. The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function. Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells. This molecular-shift appeared depending on an activation of p34Cdc2 kinase.

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Section: Discussionsupporting

confidence: 76%

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Seki

Yamashita

Nishitani

et al. 1992

MBoC

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“…Preliminary studies by Finlay and Forbes [19901 suggest the presence of 8-10 different possible NLSreceptor molecules associated with the nuclear pore. NLS-binding polypeptides, shuttling between the cytoplasm and the nucleoplasm or the nucleolus, may be present in addition to those associated with the pore [Borer et al, 1989;Yamasaki et al, 1989;Adam et al, 1989;Blobel, 1990, 1992;Lee et al, 1991;Li et al, 19921.…”

Section: Nls-binding Proteinsmentioning

confidence: 99%

Putative nuclear localization signals (NLS) in protein transcription factors

Boulikas

1994

J of Cellular Biochemistry

188

121

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We have recognized about ten distinct forms of strongly basic hexapeptides, containing at least four arginines and lysines, characteristic of nuclear proteins among all eukaryotic species, including yeast, plants, flies and mammals. These basic hexapeptides are considered to be different versions of a core nuclear localization signal, NLS. Core NLSs are present in nearly all nuclear proteins and absent from nearly all "nonassociated" cytoplasmic proteins that have been investigated. We suggest that the few ( -10%) protein factors lacking a typical NLS core peptide may enter the nucleus via their strong crosscomplexation with their protein factor partners that possess a core NLS. Those cytoplasmic proteins found to possess a NLS-like peptide are either tightly associated with cell membrane proteins or are integral components of large cytoplasmic protein complexes. On the other hand, some versions of core NLSs are found in many cell membrane proteins and secreted proteins. It is hypothesized that in these cases the N-terminal hydrophobic signal peptide of extracellular proteins and the internal hydrophobic domains of transmembrane proteins are stronger determinants for their subcellular localization. The position of core NLSs among homologous nuclear proteins may or may not be conserved; however, if lost from an homolgous site it appears elsewhere in the protein.This search provides a set of rules to our understanding of the nature of core nuclear localization signals: (1 ) Core NLS are proposed to consist most frequently of an hexapeptide with 4 arginines and lysines; ( 2 ) aspartic and glutamic acid residues as well as bulky amino acids (F, Y, W) need not to be present in this hexapeptide; (3) acidic residues and proline or glycine that break the a-helix are frequently in the flanking region of this hexapeptide stretch; (4) hydrophobic residues ought not to be present in the core NLS flanking region allowing for the NLS to be exposed on the protein. In this study we attempt to classify putative core NLS from a wealth of nuclear protein transcription factors from diverse species into several categories, and we propose additional core NLS structures yet to be experimentally verified. Key words: pore complex, transporter proteins, transcription factors, nuclear localization, nuclear proteins, basic region, cytoplasmic proteins, membrane proteins, c-Myc, c-Jun, NF-KB The nuclear envelope effectively separates transcription of genes from translation of their mRNA into proteins and allows proteins destined to function in the nucleus to pass selectively through the lumen of the nuclear pores [reviewed by Newport and Forbes, 1987; Boulikas, 19871. Pore complexes are anchored to the double nuclear membrane via the transmembrane glycoprotein gp210 [Wozniak et al., 19891 and with the aqueous channel that has an effective diameter of approximately 10 nm allow small proteins with a molecular weight inferior ~~ Abbreviations: ER, endoplasmic reticulum; aa, amino acid(s); NLS, nuclear localization signal.

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“…This binding was also dependent upon an NLS competent for nuclear import. Addition of HSA coupled to the reverse-NLS peptide (which does not support nuclear import [Adam and Gerace, 1989]), resulted in no binding (data not shown). In the presence or absence of 6xHis-Srplp and/or NLS-HSA, GST-Kap95p continued to bind Nupl16p, Nup145p, and Nupl00p (data not shown).…”

Section: Kap95p Nupll6pglfgmentioning

confidence: 99%

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

Iovine

Watkins

Wente

1995

The Journal of Cell Biology

190

201

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Abstract. Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nupll6p's role in NPC function. First, deletion mutagenesis analysis of NUPll6 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nupl00p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nupll6p's GLFG region with either Nupl00p's GLFG region or Nsplp's FXFG repetitive region abolishes the function of Nupl16p. At a semipermissive growth temperature, the cells lacking Nupl16p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nupl16p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin [3. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

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Identification of specific binding proteins for a nuclear location sequence

Cited by 217 publications

References 22 publications

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Putative nuclear localization signals (NLS) in protein transcription factors

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

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