scholarly journals Context affects nuclear protein localization in Saccharomyces cerevisiae.

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.

Context affects nuclear protein localization in Saccharomyces cerevisiae

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.

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

1987 ◽  
Vol 7 (11) ◽  
pp. 4048-4057
Author(s):  
R B Moreland ◽  
G L Langevin ◽  
R H Singer ◽  
R L Garcea ◽  
L M Hereford
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fusion Protein ◽  
Point Mutation ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Amino Acid Sequences ◽  
Nuclear Localization Sequence ◽  
Beta Galactosidase

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.

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

1987 ◽  
Vol 7 (11) ◽  
pp. 4048-4057 ◽  
Author(s):  
R B Moreland ◽  
G L Langevin ◽  
R H Singer ◽  
R L Garcea ◽  
L M Hereford
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fusion Protein ◽  
Point Mutation ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Amino Acid Sequences ◽  
Nuclear Localization Sequence ◽  
Beta Galactosidase

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.

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

2000 ◽  
Vol 353 (1) ◽  
pp. 69-77 ◽  
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.

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

1991 ◽  
Vol 280 (1) ◽  
pp. 111-116 ◽  
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.

scholarly journals Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

1997 ◽  
Vol 17 (10) ◽  
pp. 6122-6130 ◽  
Author(s):  
A W Johnson
Keyword(s):  
Fluorescent Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Cytoplasmic Rna ◽  
Localization Sequence ◽  
Green Fluorescent

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.

scholarly journals Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts

2002 ◽  
Vol 76 (7) ◽  
pp. 3145-3157 ◽  
Author(s):  
Tina M. Beachy ◽  
Sara L. Cole ◽  
Jane F. Cavender ◽  
Mary J. Tevethia
Keyword(s):  
Amino Acids ◽  
Simian Virus 40 ◽  
Suppressive Effect ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Rat Embryo ◽  
Specific Amino Acid ◽  
Antigen Fragment ◽  
Transcriptional Transactivator

ABSTRACT Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

scholarly journals Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (3) ◽  
pp. 947-957 ◽  
Author(s):  
N J Axelrod ◽  
G G Carmichael ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Evolutionary Relationship ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Yeast Cells ◽  
Upstream Activation Sequence ◽  
Beta Galactosidase ◽  
Activation Sequence

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

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