scholarly journals Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

1999 ◽  
Vol 19 (2) ◽  
pp. 1518-1525 ◽  
Author(s):  
Nilson I. T. Zanchin ◽  
David S. Goldfarb
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nucleolar Protein ◽  
Mrna Turnover ◽  
5.8S Rrna ◽  
Multistep Process ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACT NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.

scholarly journals Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits

2008 ◽  
Vol 28 (10) ◽  
pp. 3151-3161 ◽  
Author(s):  
Joshua White ◽  
Zhihua Li ◽  
Richa Sardana ◽  
Janusz M. Bujnicki ◽  
Edward M. Marcotte ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Nuclear Accumulation ◽  
Methyltransferase Activity ◽  
Late Step ◽  
Green Fluorescent

ABSTRACT BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 563-572 ◽  
Author(s):  
Valmik K Vyas ◽  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid System ◽  
Fluorescent Protein ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Green Fluorescent ◽  
Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

Biologicals ◽  
2019 ◽  
Vol 59 ◽  
pp. 56-61 ◽  
Author(s):  
Shuyun Qin ◽  
Dmitriy Volokhov ◽  
Elvira Rodionova ◽  
Christoph Wirblich ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Protein A ◽  
Fast Approach ◽  
Green Fluorescent

scholarly journals Multiple Adhesin-Like Functions of Xanthomonas oryzae pv. oryzae Are Involved in Promoting Leaf Attachment, Entry, and Virulence on Rice

2009 ◽  
Vol 22 (1) ◽  
pp. 73-85 ◽  
Author(s):  
Amit Das ◽  
Nandini Rangaraj ◽  
Ramesh V. Sonti
Keyword(s):  
Bacterial Adhesion ◽  
Bacterial Blight ◽  
Fluorescent Protein ◽  
Protein A ◽  
Disease Process ◽  
Xanthomonas Oryzae ◽  
Type Iv ◽  
Genes Encoding ◽  
Green Fluorescent

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. We have used enhanced green fluorescent protein-tagged X. oryzae pv. oryzae cells in conjunction with confocal microscopy to monitor the role of several adhesin-like functions in bacterial adhesion to leaf surface and early stages of leaf entry. Mutations in genes encoding either the Xanthomonas adhesin-like protein A (XadA) or its paralog, Xanthomonas adhesin-like protein B (XadB), as well as the X. oryzae pv. oryzae homolog of Yersinia autotransporter-like protein H (YapH), exhibit deficiencies in leaf attachment or entry. A mutation in the X. oryzae pv. oryzae pilQ gene, which is predicted to encode the type IV pilus secretin, appears to have no effect on leaf attachment or entry. The xadA– mutant is deficient in the ability to cause disease following surface inoculation while the XadB, YapH, and PilQ functions are less important than XadA for this process. The xadA– and xadB– mutants have no effect on virulence following wound inoculation whereas the yapH– and pilQ– mutants are always virulence deficient following wound inoculation. Overall, these results indicate that multiple adhesin-like functions are involved in promoting virulence of X. oryzae pv. oryzae, with preferential involvement of individual functions at different stages of the disease process.

Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides

2007 ◽  
Vol 35 (4) ◽  
pp. 797-801 ◽  
Author(s):  
J.A. Gomez ◽  
V. Gama ◽  
T. Yoshida ◽  
W. Sun ◽  
P. Hayes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Protein A ◽  
Cellular Damage ◽  
Toxic Drug ◽  
Cell Penetrating ◽  
Low Toxicity ◽  
Repair Factor ◽  
Green Fluorescent

We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4°C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.

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