Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Janet M Murray ◽  
Douglas I Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fluorescent Protein ◽  
Amino Acid Identity ◽  
Negative Regulator ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Isolation And Characterization ◽  
Nucleotide Exchange Factor ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.

scholarly journals Isolation and Characterization of YlBEM1, a Gene Required for Cell Polarization and Differentiation in the Dimorphic Yeast Yarrowia lipolytica

2002 ◽  
Vol 1 (4) ◽  
pp. 526-537 ◽  
Author(s):  
Cleofe A. R. Hurtado ◽  
Richard A. Rachubinski
Keyword(s):  
Yarrowia Lipolytica ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Pathogenic Fungi ◽  
Cell Polarization ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Dimorphic Yeast ◽  
Green Fluorescent

ABSTRACT The ability to switch between a unicellular yeast form and different filamentous forms (fungal dimorphism) is an important attribute of most pathogenic fungi. Dimorphism involves a series of events that ultimately result in dramatic changes in the polarity of cell growth in response to environmental factors. We have isolated and characterized YlBEM1, a gene encoding a protein of 639 amino acids that is essential for the yeast-to-hypha transition in the yeast Yarrowia lipolytica and whose transcription is significantly increased during this event. Cells with deletions of YlBEM1 are viable but show substantial alterations in morphology, disorganization of the actin cytoskeleton, delocalization of cortical actin and chitin deposition, multinucleation, and loss of mating ability, thus pointing to a major role for YlBEM1 in the regulation of cell polarity and morphogenesis in this fungus. This role is further supported by the localization of YlBem1p, which, like cortical actin, appears to be particularly abundant at sites of growth of yeast, hyphal, and pseudohyphal cells. In addition, the potential involvement of YlBem1p in septum formation and/or cytokinesis is suggested by the concentration of a green fluorescent protein-tagged version of this protein at the mother-bud neck during the last stages of cell division. Interestingly, overexpression of MHY1, YlRAC1, or YlSEC31, three genes involved in filamentous growth of Y. lipolytica, induced hyphal growth of bem1 null mutant cells.

scholarly journals Yeast Ysl2p, Homologous to Sec7 Domain Guanine Nucleotide Exchange Factors, Functions in Endocytosis and Maintenance of Vacuole Integrity and Interacts with the Arf-Like Small GTPase Arl1p

2002 ◽  
Vol 22 (13) ◽  
pp. 4914-4928 ◽  
Author(s):  
Alexandra Jochum ◽  
David Jackson ◽  
Heinz Schwarz ◽  
Rüdiger Pipkorn ◽  
Birgit Singer-Krüger
Keyword(s):  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Small Gtpase ◽  
Temperature Sensitive ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Green Fluorescent ◽  
Vacuolar Protein

ABSTRACT We previously described the isolation of ysl2-1 due to its genetic interaction with Δypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and α-factor and exhibit vacuole fragmentation directly upon a shift to 37°C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Δarl1 cells are defective in endocytic transport and in vacuolar protein sorting.

scholarly journals Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin, a Ubiquitous Actin Monomer-sequestering Protein

1998 ◽  
Vol 142 (3) ◽  
pp. 723-733 ◽  
Author(s):  
Bruce L. Goode ◽  
David G. Drubin ◽  
Pekka Lappalainen
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Green Fluorescent

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

scholarly journals Cytokinins Stimulate Plasmodesmatal Transport in Leaves

2021 ◽  
Vol 12 ◽  
Author(s):  
Wilson Horner ◽  
Jacob O. Brunkard
Keyword(s):  
Cell Walls ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Plant Biology ◽  
Negative Regulator ◽  
Cytokinin Signaling ◽  
Dynamic Changes ◽  
Cytokinin Receptors ◽  
Green Fluorescent ◽  
Development Regulation

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1–4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae

1996 ◽  
Vol 109 (9) ◽  
pp. 2311-2318 ◽  
Author(s):  
N. Nakashima ◽  
N. Hayashi ◽  
E. Noguchi ◽  
T. Nishimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Importin Alpha ◽  
Nucleotide Exchange Factor ◽  
Cold Sensitive ◽  
Gtpase Cycle

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

scholarly journals Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase

2003 ◽  
Vol 371 (2) ◽  
pp. 533-540 ◽  
Author(s):  
Shary N. SHELTON ◽  
Barbara BARYLKO ◽  
Derk D. BINNS ◽  
Bruce F. HORAZDOVSKY ◽  
Joseph P. ALBANESI ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Distinct Type ◽  
Type Ii ◽  
Yeast Saccharomyces Cerevisiae ◽  
Molar Ratios ◽  
Ionic Detergent ◽  
Km Value ◽  
Green Fluorescent ◽  
Vacuolar Membranes

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100min−1 were obtained at the PI/Triton X-100 ratio of 1:5. The Km value for ATP was 266μM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p–green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 212
Author(s):  
C. A. Pinzon ◽  
M. Snyder ◽  
J. Pryor ◽  
B. Thompson ◽  
M. Golding ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Muscle Mass ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Negative Regulator ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

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