scholarly journals Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi

2004 ◽  
Vol 378 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Maximiliano A. D'ANGELO ◽  
Santiago SANGUINETI ◽  
Jeffrey M. REECE ◽  
Lutz BIRNBAUMER ◽  
Héctor N. TORRES ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Subcellular Localization ◽  
Laser Scanning ◽  
Phosphodiesterase Inhibitor ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Sodium Cholate ◽  
Confocal Laser ◽  
Acid Protein ◽  
Amino Acid Protein

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway

1993 ◽  
Vol 104 (3) ◽  
pp. 671-683 ◽  
Author(s):  
A. Schweizer ◽  
M. Ericsson ◽  
T. Bachi ◽  
G. Griffiths ◽  
H.P. Hauri
Keyword(s):  
Membrane Protein ◽  
Laser Scanning ◽  
Protein Transport ◽  
Brefeldin A ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Confocal Laser ◽  
Similar Degree ◽  
Fractionation Procedure ◽  
Intermediate Compartment

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis

1999 ◽  
Vol 112 (22) ◽  
pp. 4031-4039 ◽  
Author(s):  
M. Koike ◽  
T. Awaji ◽  
M. Kataoka ◽  
G. Tsujimoto ◽  
T. Kartasova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Laser Scanning ◽  
Growth Regulation ◽  
Strand Break ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Metaphase Chromosomes ◽  
Interphase Cells ◽  
Ku Protein

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Paige Lacy ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
Juan R. Velazquez ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Human Peripheral Blood ◽  
Subcellular Fractionation ◽  
Secretory Vesicle ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf ◽  
Ifn Γ

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

scholarly journals Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6775-6787 ◽  
Author(s):  
Doug J. Bartels ◽  
David A. Mitchell ◽  
Xiangwen Dong ◽  
Robert J. Deschenes
Keyword(s):  
Subcellular Localization ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Growth Defect ◽  
Loss Of Function ◽  
Reading Frame ◽  
Rich Domain ◽  
Carboxy Terminal ◽  
The Relationship ◽  
Yeast Genetic

ABSTRACT Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependentRAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactiveRAS2(V19) allele. Ras2 proteins expressed in anerf2Δ strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.

Carbonic anhydrases IV and IX: subcellular localization and functional role in mouse skeletal muscle

2008 ◽  
Vol 294 (2) ◽  
pp. C402-C412 ◽  
Author(s):  
Renate J. Scheibe ◽  
Karsten Mundhenk ◽  
Tilman Becker ◽  
Janine Hallerdei ◽  
Abdul Waheed ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Subcellular Localization ◽  
Laser Scanning ◽  
Muscle Fibers ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mouse Skeletal Muscle ◽  
Ca Ix ◽  
Skeletal Muscle Fibers

The subcellular localization of carbonic anhydrase (CA) IV and CA IX in mouse skeletal muscle fibers has been studied immunohistochemically by confocal laser scanning microscopy. CA IV has been found to be located on the plasma membrane as well as on the sarcoplasmic reticulum (SR) membrane. CA IX is not localized in the plasma membrane but in the region of the t-tubular (TT)/terminal SR membrane. CA IV contributes 20% and CA IX 60% to the total CA activity of SR membrane vesicles isolated from mouse skeletal muscles. Our aim was to examine whether SR CA IV and TT/SR CA IX affect muscle contraction. Isolated fiber bundles of fast-twitch extensor digitorum longus and slow-twitch soleus muscle from mouse were investigated for isometric twitch and tetanic contractions and by a fatigue test. The muscle functions of CA IV knockout (KO) fibers and of CA IX KO fibers do not differ from the function of wild-type (WT) fibers. Muscle function of CA IV/XIV double KO mice unexpectedly shows a decrease in rise and relaxation time and in force of single twitches. In contrast, the CA inhibitor dorzolamide, whether applied to WT or to double KO muscle fibers, leads to a significant increase in rise time and force of twitches. It is concluded that the function of mouse skeletal muscle fibers expressing three membrane-associated CAs, IV, IX, and XIV, is not affected by the lack of one isoform but is possibly affected by the lack of all three CAs, as indicated by the inhibition studies.

scholarly journals Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

2000 ◽  
Vol 149 (3) ◽  
pp. 623-634 ◽  
Author(s):  
George Hausmann ◽  
Lorraine A. O'Reilly ◽  
Rosemary van Driel ◽  
Jennifer G. Beaumont ◽  
Andreas Strasser ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Laser Scanning ◽  
Subcellular Fractionation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Cytoplasmic Localization ◽  
In Vitro Activation

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease–activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2–like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2–like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

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