scholarly journals Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect

2007 ◽  
Vol 407 (2) ◽  
pp. 161-170 ◽  
Author(s):  
Jianmin Fang ◽  
Peter Rohloff ◽  
Kildare Miranda ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Immunogold Electron Microscopy ◽  
Protonmotive Force ◽  
Morphological Alterations ◽  
A Cell ◽  
Green Fluorescent ◽  
And Function

Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H+-PPase (vacuolar H+-pyrophosphatase), a marker for acidocalcisomes. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H+ uptake and Ca2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. We propose that the PPi-driven H+ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H+ and Ca2+ content, and decreased synthesis of poly P. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis.

scholarly journals Analysis of membrane proteins localizing to the inner nuclear envelope in living cells

2016 ◽  
Vol 215 (4) ◽  
pp. 575-590 ◽  
Author(s):  
Christine J. Smoyer ◽  
Santharam S. Katta ◽  
Jennifer M. Gardner ◽  
Lynn Stoltz ◽  
Scott McCroskey ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Transmembrane Protein ◽  
Protein Composition ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Green Fluorescent ◽  
And Function

Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.

scholarly journals Stabilization of Polar Localization of a Chemoreceptor via Its Covalent Modifications and Its Communication with a Different Chemoreceptor

2005 ◽  
Vol 187 (22) ◽  
pp. 7647-7654 ◽  
Author(s):  
Daisuke Shiomi ◽  
Satomi Banno ◽  
Michio Homma ◽  
Ikuro Kawagishi
Keyword(s):  
Histidine Kinase ◽  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Molecular Assembly ◽  
Covalent Modification ◽  
Polar Localization ◽  
Receptor Localization ◽  
A Cell ◽  
Covalent Modifications ◽  
Green Fluorescent

ABSTRACT In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

scholarly journals The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

2014 ◽  
Vol 14 (3) ◽  
pp. 206-215 ◽  
Author(s):  
Steven Sykes ◽  
Anthony Szempruch ◽  
Stephen Hajduk
Keyword(s):  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Krebs Cycle ◽  
Content Type ◽  
African Trypanosomes ◽  
Atp Production ◽  
Cycle Enzyme ◽  
A Cell ◽  
Direct Localization ◽  
Green Fluorescent

ABSTRACT α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei . The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei . These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei .

scholarly journals Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

1999 ◽  
Vol 146 (2) ◽  
pp. 333-344 ◽  
Author(s):  
Chavela M. Carr ◽  
Eric Grote ◽  
Mary Munson ◽  
Frederick M. Hughson ◽  
Peter J. Novick
Keyword(s):  
Fluorescent Protein ◽  
Snare Complex ◽  
The Other ◽  
Complex Assembly ◽  
Vesicle Docking ◽  
Neuronal Protein ◽  
The Core ◽  
Target Membrane ◽  
Green Fluorescent ◽  
And Function

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

Abstract 3880: GRK2 Inhibition Prevents Development Of Cardiac Insulin Resistance And Impaired Glucose Uptake In Post Ischemic Heart Failure

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Michele Ciccarelli ◽  
Giuseppe Rengo ◽  
Kurt Chuprun ◽  
Gaetano Santulli ◽  
Bruno Trimarco ◽  
...  
Keyword(s):  
Heart Failure ◽  
Glucose Uptake ◽  
Fluorescent Protein ◽  
Sprague Dawley ◽  
Myocardial Glucose Uptake ◽  
Akt Phosphorylation ◽  
Neonatal Cardiomyocytes ◽  
Sprague Dawley Rats ◽  
Green Fluorescent ◽  
And Function

The beta adrenergic receptor (βAR) kinase, GRK2, is upregulated and participates to the evolution of heart failure (HF) through downregulation and desensitization of βARs. Recent studies showed that this molecule affects insulin signaling and reduce glucose uptake in hepatocytes and adipocytes. We hypothesized that in HF, GRK2 reduces cardiac performance also through inhibition of cardiac glucose metabolism. In 12 week old Sprague/Dawley rats, we measured cardiac glucose uptake by PET 3 days, 3 and 6 weeks after myocardial infarction (MI). Function and cardiac dimensions were measured by echocardiography. We observed that glucose uptake was reduced in animal post-MI at 3 and 6 weeks respect to healthy animals (3 rd week: 1.3±0.22 vs 2.1±0.3; 6 th week: 1±0.1 vs 2.4±0.2, ml/min/g, p<0.05). No difference was observed in glucose uptake acutely after surgery. Echo showed cardiac dilation and reduced function at 6 weeks (LVD: 9.2± 0.3 vs 7.2± 0.4 mm; EF: 42%±1.1 vs 66%±2.2, p<0.05, Sham vs MI). To inhibit GRK2 in the heart during post-ischemic HF, we delivered the GRK2 inhibitor βARKct by adeno-associated type 6 virus (AAV6) to the left ventricle before induction of the MI. As a control we treated rats with AAV6 encoding for the green fluorescent protein (GFP). Cardiac dilation and function were preserved after 6 weeks post MI in AAV6 βARKct respect to AAV6GFP rats (LVD: 7.73 ±0.25 vs 9.9 ±0.8 mm; EF: 55%±2.25 vs 44%±2, p<0.05). Glucose uptake was better preserved in AAV6βARKct rats after 3 and 6 weeks post MI respect to AAV6GFP group (3rd week: 2.3±0.3 vs 1.2±0.2; 6th week: 1.8±0.2 vs 1.1±0.05, ml/min/g, p<0.05). Since Akt mediates most of the anabolic effects of insulin in cells, we evaluated the effects of GRK2 overexpression by adenovirus (ADGRK2) in neonatal cardiomyocytes (NRVMs) on Akt phosphorylation later on insulin stimulation (ins, 10 – 6 M). As control we induced overexpression of GFP by adenovirus (ADGFP). We observed reduced activation of Akt in presence of GRK2 overexpression as compared to the ADGFP treated cells (1.2±0.2- vs. 3.5±0.4- fold activation over basal, p<0.05). Our data show that post MI, impaired glucose extraction precedes development of HF, and that early GRK2 inhibition prevents impaired myocardial glucose uptake and HF development.

scholarly journals Two Point Mutations Produce Infectious Retrovirus Bearing a Green Fluorescent Protein-SU Fusion Protein

2001 ◽  
Vol 75 (23) ◽  
pp. 11881-11885 ◽  
Author(s):  
Krishnakumar Kizhatil ◽  
Adam Gromley ◽  
Lorraine M. Albritton
Keyword(s):  
Green Fluorescent Protein ◽  
Envelope Protein ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fundamental Problem ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Slight Reduction ◽  
Virus Envelope ◽  
Green Fluorescent

ABSTRACT Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.

scholarly journals Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

2015 ◽  
Vol 113 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Marie-Aude Plamont ◽  
Emmanuelle Billon-Denis ◽  
Sylvie Maurin ◽  
Carole Gauron ◽  
Frederico M. Pimenta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Activation Mechanism ◽  
Photoactive Yellow Protein ◽  
Reversible Binding ◽  
A Cell ◽  
Rapid Labeling ◽  
Green Fluorescent

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

scholarly journals Deposition and Function of Histone H3 Variants in Tetrahymena thermophila

2006 ◽  
Vol 26 (20) ◽  
pp. 7719-7730 ◽  
Author(s):  
Bowen Cui ◽  
Yifan Liu ◽  
Martin A. Gorovsky
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Histone H3 ◽  
Repair Synthesis ◽  
Nucleosome Density ◽  
Sexual Progeny ◽  
Green Fluorescent ◽  
Dna Repair Synthesis ◽  
And Function

ABSTRACT In Tetrahymena, HHT1 and HHT2 genes encode the same major histone H3; HHT3 and HHT4 encode similar minor H3 variants (H3s), H3.3 and H3.4. Green fluorescent protein (GFP)-tagged H3 is deposited onto chromatin through a DNA replication-coupled (RC) pathway. GFP-tagged H3.3 and H3.4 can be deposited both by a transcription-associated, replication-independent (RI) pathway and also weakly by an RC pathway. Although both types of H3s can be deposited by the RC pathway, DNA repair synthesis associated with meiotic recombination utilizes H3 specifically. The regions distinguishing H3 and H3.3 for their deposition pathways were identified. RC major H3 is not essential. Cells can grow without major H3 if the minor H3s are expressed at high levels. Surprisingly, cells lacking RI H3s are also viable and maintain normal nucleosome density at a highly transcribed region. The RC H3 is not detectably deposited by the RI pathway, even when there are no RI H3s available, indicating that transcription-associated RI H3 deposition is not essential for transcription. Minor H3s are also required to produce viable sexual progeny and play an unexpected role in the germ line micronuclei late in conjugation that is unrelated to transcription.

scholarly journals MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis

2020 ◽  
Vol 71 (16) ◽  
pp. 4877-4889
Author(s):  
Jie-Yang Lu ◽  
Shuang-Xi Xiong ◽  
Wenzhe Yin ◽  
Xiao-Dong Teng ◽  
Yue Lou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Pollen Wall ◽  
Coat Proteins ◽  
Related Family ◽  
Regulatory Cascade ◽  
Pollen Coat ◽  
Green Fluorescent ◽  
High Level ◽  
And Function

Abstract Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen–stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP–oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

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