Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

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Context affects nuclear protein localization in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.384 ◽

1989 ◽

Vol 9 (2) ◽

pp. 384-389 ◽

Cited By ~ 61

Author(s):

M Nelson ◽

P Silver

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Nuclear Localization ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Single Amino Acid ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

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Context affects nuclear protein localization in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.384-389.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 384-389

Author(s):

M Nelson ◽

P Silver

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Nuclear Localization ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Single Amino Acid ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

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Analysis of mutations in the putative nuclear localization sequence of interleukin-1β

Biochemical Journal ◽

10.1042/bj2800111 ◽

1991 ◽

Vol 280 (1) ◽

pp. 111-116 ◽

Cited By ~ 21

Author(s):

S Grenfell ◽

N Smithers ◽

S Witham ◽

A Shaw ◽

P Graber ◽

...

Keyword(s):

Nuclear Localization ◽

Interleukin 1 ◽

Simian Virus 40 ◽

T Antigen ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

Target Cells ◽

Wild Type ◽

Receptor Mediated Endocytosis ◽

Localization Sequence

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210→Ala), M50 (Lys211→Ala) and M51 (Pro208→Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

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The Protein Kinase CK2 Site (Ser111/112) Enhances Recognition of the Simian Virus 40 Large T-antigen Nuclear Localization Sequence by Importin

Journal of Biological Chemistry ◽

10.1074/jbc.272.27.17191 ◽

1997 ◽

Vol 272 (27) ◽

pp. 17191-17195 ◽

Cited By ~ 175

Author(s):

Stefan Hübner ◽

Chong-Yun Xiao ◽

David A. Jans

Keyword(s):

Protein Kinase ◽

Nuclear Localization ◽

Protein Kinase Ck2 ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Nuclear Localization Sequence ◽

Large T Antigen ◽

Localization Sequence ◽

Kinase Ck2

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Multiple regions of NSR1 are sufficient for accumulation of a fusion protein within the nucleolus.

The Journal of Cell Biology ◽

10.1083/jcb.123.5.1081 ◽

1993 ◽

Vol 123 (5) ◽

pp. 1081-1091 ◽

Cited By ~ 31

Author(s):

C Yan ◽

T Mélèse

Keyword(s):

Fusion Protein ◽

Nuclear Localization ◽

Ribosome Biogenesis ◽

Nuclear Localization Sequence ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rich Region ◽

Rna Recognition Motifs ◽

Recognition Motifs ◽

Beta Galactosidase

NSR1, a 67-kD nucleolar protein, was originally identified in our laboratory as a nuclear localization signal binding protein, and has subsequently been found to be involved in ribosome biogenesis. NSR1 has three regions: an acidic/serine-rich NH2 terminus, two RNA recognition motifs, and a glycine/arginine-rich COOH terminus. In this study we show that NSR1 itself has a bipartite nuclear localization sequence. Deletion of either basic amino acid stretch results in the mislocation of NSR1 to the cytoplasm. We further demonstrate that either of two regions, the NH2 terminus or both RNA recognition motifs, are sufficient to localize a bacterial protein, beta-galactosidase, to the nucleolus. Intensive deletion analysis has further defined a specific acidic/serine-rich region within the NH2 terminus as necessary for nucleolar accumulation rather than nucleolar targeting. In addition, deletion of either RNA recognition motif or point mutations in one of the RNP consensus octamers results in the mislocalization of a fusion protein within the nucleus. Although the glycine/arginine-rich region in the COOH terminus is not sufficient to bring beta-galactosidase to the nucleolus, our studies show that this domain is necessary for nucleolar accumulation when an RNP consensus octamer in one of the RNA recognition motifs is mutated. Our findings are consistent with the notion that nucleolar localization is a result of the binding interactions of various domains of NSR1 within the nucleolus rather than the presence of a specific nucleolar targeting signal.

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Specific Amino Acid Residues in the Basic Helix-Loop-Helix Domain of SRC-3 Are Essential for Its Nuclear Localization and Proteasome-Dependent Turnover

Molecular and Cellular Biology ◽

10.1128/mcb.00336-06 ◽

2006 ◽

Vol 27 (4) ◽

pp. 1296-1308 ◽

Cited By ~ 31

Author(s):

Chao Li ◽

Ray-Chang Wu ◽

Larbi Amazit ◽

Sophia Y. Tsai ◽

Ming-Jer Tsai ◽

...

Keyword(s):

Amino Acid ◽

Nuclear Localization ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Amino Acid Residues ◽

Transcriptional Coactivator ◽

Basic Helix Loop Helix ◽

Bhlh Domain ◽

Helix Loop Helix

ABSTRACT SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1 is a primary transcriptional coactivator for the estrogen receptor. Here we report that deletion of the SRC-3 basic helix-loop-helix (bHLH) domain blocks its proteasome-dependent turnover. We further identified two residues (K17 and R18) in the SRC-3 bHLH domain that are essential for its stability. Moreover, we found that the bHLH domain contains a bipartite nuclear localization signal (NLS). SRC-3 NLS mutants block its translocation into the nucleus, and this correlates with its insensitivity to proteasome-dependent turnover. SRC-3 shows a time-dependent decay in the presence of cycloheximide which is not apparent for the cytoplasmic mutant. Fusion of a simian virus 40 T antigen NLS to the cytoplasmic localized SRC-3 mutant drives it back into the nucleus and restores its proteasomal sensitivity. In addition, the cytoplasmic mutants are inactive for transcriptional coactivation and cancer cell growth. Taken together, our data indicate that proteasome-dependent turnover of SRC-3 occurs in the nucleus and that two amino acid residues in the bHLH domain provide a signal for its nuclear localization and proteasome-dependent degradation as well as for regulation of SRC-3 transcriptional coactivator capacity.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

Biochemical Journal ◽

10.1042/bj1310485 ◽

1973 ◽

Vol 131 (3) ◽

pp. 485-498 ◽

Cited By ~ 113

Author(s):

R. P. Ambler ◽

Margaret Wynn

Keyword(s):

Amino Acids ◽

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Cytochrome C ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Mitochondrial Cytochrome ◽

Cytochromes C ◽

Lending Library ◽

Single Peptide

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

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Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16

Biochemical Journal ◽

10.1042/bj3530069 ◽

2000 ◽

Vol 353 (1) ◽

pp. 69-77 ◽

Cited By ~ 24

Author(s):

Lyndall J. BRIGGS ◽

Ricky W. JOHNSTONE ◽

Rachel M. ELLIOT ◽

Chong-Yun XIAO ◽

Michelle DAWSON ◽

...

Keyword(s):

Protein Kinase ◽

Nuclear Localization ◽

Nuclear Import ◽

Protein Kinase Ck2 ◽

Protein Import ◽

Simian Virus 40 ◽

Simian Virus ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

Kinase Ck2

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

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