scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals OASTL-A1 functions as a cytosolic cysteine synthase and affects arsenic tolerance in rice

2020 ◽  
Vol 71 (12) ◽  
pp. 3678-3689 ◽  
Author(s):  
Chengcheng Wang ◽  
Lihua Zheng ◽  
Zhong Tang ◽  
Shengkai Sun ◽  
Jian Feng Ma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Tissue Expression ◽  
Protein Fusion ◽  
Outer Cortex ◽  
Cysteine Synthase ◽  
Arsenic Tolerance ◽  
Tissue Expression Analysis ◽  
Green Fluorescent

Abstract Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O-acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O-acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O-acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

scholarly journals Yeast Exocytic v-SNAREs Confer Endocytosis

2000 ◽  
Vol 11 (10) ◽  
pp. 3629-3643 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Daphne Chapman-Shimshoni ◽  
Selena Trajkovic ◽  
Jeffrey E. Gerst
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Metabolic Labeling ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Direct Role ◽  
Endosomal Transport ◽  
Sensitive Allele ◽  
Green Fluorescent

In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1(SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that bothSNC and temperature-shiftedSNC1ala43yeast are deficient in their ability to deliver the soluble dye FM4–64 to the vacuole. Under conditions in which vesicles accumulate, FM4–64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, α-factor–stimulated endocytosis of the α-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1ala43was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.

scholarly journals Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

2009 ◽  
Vol 191 (9) ◽  
pp. 3149-3161 ◽  
Author(s):  
Suzanne R. Lybarger ◽  
Tanya L. Johnson ◽  
Miranda D. Gray ◽  
Aleksandra E. Sikora ◽  
Maria Sandkvist
Keyword(s):  
Vibrio Cholerae ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

scholarly journals The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

1999 ◽  
Vol 146 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Alexey Khodjakov ◽  
Conly L. Rieder
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent ◽  
Dynamic Exchange ◽  
Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals ATP-Dependent Localization of the Herpes Simplex Virus Capsid Protein VP26 to Sites of Procapsid Maturation

2000 ◽  
Vol 74 (3) ◽  
pp. 1468-1476 ◽  
Author(s):  
Jung Hee I. Chi ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Cold Sensitivity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Protein Fusion ◽  
Capsid Shell ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutanttsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.

scholarly journals A Nuclear 3′-5′ Exonuclease Involved in mRNA Degradation Interacts with Poly(A) Polymerase and the hnRNA Protein Npl3p

2000 ◽  
Vol 20 (2) ◽  
pp. 604-616 ◽  
Author(s):  
Karina T. D. Burkard ◽  
J. Scott Butler
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Genetic Selection ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Mrna Binding Protein ◽  
Cold Sensitive ◽  
Green Fluorescent ◽  
Mrna Binding ◽  
Mrna Polyadenylation

ABSTRACT Inactivation of poly(A) polymerase (encoded by PAP1) inSaccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)+ mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors,rrp6-1 and rrp6-2, as well as a deletion ofRRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)+mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3′-5′ exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3′-5′ exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)+ mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.

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