Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

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.

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Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

Microbiology ◽

10.1099/mic.0.27692-0 ◽

2005 ◽

Vol 151 (3) ◽

pp. 999-1012 ◽

Cited By ~ 16

Author(s):

Dirk-Jan Scheffers

Keyword(s):

Bacillus Subtilis ◽

Green Fluorescent Protein ◽

Binding Proteins ◽

Cytoplasmic Membrane ◽

Fluorescent Protein ◽

Spore Formation ◽

The Other ◽

Spore Development ◽

Penicillin Binding Proteins ◽

Green Fluorescent

During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.

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Changes in the Min Oscillation Pattern before and after Cell Birth

Journal of Bacteriology ◽

10.1128/jb.00364-10 ◽

2010 ◽

Vol 192 (16) ◽

pp. 4134-4142 ◽

Cited By ~ 24

Author(s):

Jennifer R. Juarez ◽

William Margolin

Keyword(s):

Cell Division ◽

Fluorescent Protein ◽

Time Lapse ◽

The Other ◽

Cell Pole ◽

Daughter Cells ◽

Division Site ◽

Before And After ◽

Dividing Cells ◽

Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

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

Circulation ◽

10.1161/circ.118.suppl_18.s_496-d ◽

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.

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Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.00588.2003 ◽

2004 ◽

Vol 287 (5) ◽

pp. C1366-C1374 ◽

Cited By ~ 21

Author(s):

Julie A. Nicoletta ◽

Jonathan J. Ross ◽

Guangmu Li ◽

Qingzhang Cheng ◽

Jonathon Schwartz ◽

...

Keyword(s):

Fluorescent Protein ◽

Apical Membrane ◽

Collecting Duct ◽

Snare Complex ◽

Glutathione S Transferase ◽

Syntaxin 1A ◽

Inner Medullary Collecting Duct ◽

Medullary Collecting Duct ◽

Reduced Rate ◽

Green Fluorescent

Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.

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Deposition and Function of Histone H3 Variants in Tetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.01139-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7719-7730 ◽

Cited By ~ 47

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.

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

Journal of Experimental Botany ◽

10.1093/jxb/eraa219 ◽

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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Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

Biochemical Journal ◽

10.1042/bj3480119 ◽

2000 ◽

Vol 348 (1) ◽

pp. 119-128 ◽

Cited By ~ 16

Author(s):

Madeleine TOUTANT ◽

Jeanne-Marie STUDLER ◽

Ferran BURGAYA ◽

Alicia COSTA ◽

Pascal EZAN ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Fluorescent Protein ◽

Focal Adhesions ◽

Src Family Kinases ◽

Critical Residue ◽

Green Fluorescent ◽

And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

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Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽

10.1210/en.2009-0881 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5626-5632 ◽

Cited By ~ 38

Author(s):

Tamar Alon ◽

Ligang Zhou ◽

Cristian A. Pérez ◽

Alastair S. Garfield ◽

Jeffrey M. Friedman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Brain Regions ◽

Ingestive Behavior ◽

Physiological Processes ◽

Functional Relevance ◽

Green Fluorescent ◽

And Function ◽

The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

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Characterization of the mononuclear phagocyte system in zebrafish

Blood ◽

10.1182/blood-2010-11-321448 ◽

2011 ◽

Vol 117 (26) ◽

pp. 7126-7135 ◽

Cited By ~ 116

Author(s):

Valerie Wittamer ◽

Julien Y. Bertrand ◽

Patrick W. Gutschow ◽

David Traver

Keyword(s):

Fluorescent Protein ◽

Transgenic Animals ◽

Regulatory Elements ◽

Mononuclear Phagocyte ◽

Mononuclear Phagocytes ◽

Reporter Line ◽

Transgenic Lines ◽

Green Fluorescent ◽

And Function ◽

Antigen Presenting

Abstract The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFPhi; cd45:DsRedhi cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

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

The Journal of Cell Biology ◽

10.1083/jcb.201607043 ◽

2016 ◽

Vol 215 (4) ◽

pp. 575-590 ◽

Cited By ~ 40

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.

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