scholarly journals Tortoise, a Novel Mitochondrial Protein, Is Required for Directional Responses of Dictyostelium in Chemotactic Gradients

2001 ◽  
Vol 152 (3) ◽  
pp. 621-632 ◽  
Author(s):  
Saskia van Es ◽  
Deborah Wessels ◽  
David R. Soll ◽  
Jane Borleis ◽  
Peter N. Devreotes
Keyword(s):  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Wild Type ◽  
Cytosolic Ph ◽  
Spatial Gradients ◽  
Similar Phenotype ◽  
Green Fluorescent ◽  
Triton X ◽  
Shape Changes

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA− partially restores chemotaxis, whereas overexpression of TorA in mek1− does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA–green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

scholarly journals Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase

2017 ◽  
Vol 37 (23) ◽  
Author(s):  
Nadav Sorek ◽  
Limor Poraty ◽  
Hasana Sternberg ◽  
Ella Buriakovsky ◽  
Einat Bar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Wild Type ◽  
Content Type ◽  
Lipid Modifications ◽  
Membrane Partitioning ◽  
Acyl Lipids ◽  
Detergent Resistant Membranes ◽  
Green Fluorescent ◽  
Triton X ◽  
Activation Status

ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes.

Characterization of the promoter activity of a poxvirus conserved element

2008 ◽  
Vol 54 (6) ◽  
pp. 483-488
Author(s):  
Heather E. Eaton ◽  
Julie Metcalf ◽  
Craig R. Brunetti
Keyword(s):  
Fluorescent Protein ◽  
Promoter Sequence ◽  
Promoter Strength ◽  
Wild Type ◽  
Sequence Element ◽  
Promoter Function ◽  
Conserved Sequence ◽  
Polymerase Chain ◽  
Green Fluorescent

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.

scholarly journals Activation Status-Coupled Transient S Acylation Determines MembranePartitioning of a Plant Rho-Related GTPase

2007 ◽  
Vol 27 (6) ◽  
pp. 2144-2154 ◽  
Author(s):  
Nadav Sorek ◽  
Limor Poraty ◽  
Hasana Sternberg ◽  
Enat Bar ◽  
Efraim Lewinsohn ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Wild Type ◽  
Lipid Modifications ◽  
Gtp Binding ◽  
Acyl Lipids ◽  
Detergent Resistant Membranes ◽  
Green Fluorescent ◽  
Triton X ◽  
The Relationship ◽  
Activation Status

ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His6-GFP-Atrop6CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His6-GFP-Atrop6CAmS156 in which cysteine156 was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

2000 ◽  
Vol 11 (11) ◽  
pp. 3873-3883 ◽  
Author(s):  
Maryse Bailly ◽  
Jeffrey Wyckoff ◽  
Boumediene Bouzahzah ◽  
Ross Hammerman ◽  
Vonetta Sylvestre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Epidermal Growth ◽  
Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

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