scholarly journals The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

2008 ◽  
Vol 19 (11) ◽  
pp. 4888-4899 ◽  
Author(s):  
Laura A. Schroder ◽  
Michael V. Ortiz ◽  
William A. Dunn
Keyword(s):  
Pichia Pastoris ◽  
Membrane Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Guanosine Triphosphate ◽  
Guanosine Diphosphate ◽  
Mutant Forms ◽  
Negative Mutant

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

scholarly journals Functional Expression of Human PP2Ac in Yeast Permits the Identification of Novel C-terminal and Dominant-negative Mutant Forms

1999 ◽  
Vol 274 (34) ◽  
pp. 24038-24046 ◽  
Author(s):  
David R. H. Evans ◽  
Timothy Myles ◽  
Jan Hofsteenge ◽  
Brian A. Hemmings
Keyword(s):  
Functional Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Forms ◽  
Negative Mutant

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility

2004 ◽  
Vol 279 (44) ◽  
pp. 46122-46128 ◽  
Author(s):  
Indira Neeli ◽  
Zhimin Liu ◽  
Nagadhara Dronadula ◽  
Z. Alex Ma ◽  
Gadiparthi N. Rao
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Dominant Negative ◽  
Platelet Derived Growth Factor ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Cytosolic Phospholipase ◽  
Negative Mutant

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A2(cPLA2). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA2in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA2prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA2rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA2signaling axis.

scholarly journals Role of EGR-1 in thapsigargin-inducible apoptosis in the melanoma cell line A375-C6.

1995 ◽  
Vol 15 (11) ◽  
pp. 6262-6272 ◽  
Author(s):  
S Muthukkumar ◽  
P Nair ◽  
S F Sells ◽  
N G Maddiwar ◽  
R J Jacob ◽  
...  
Keyword(s):  
Actinomycin D ◽  
Interleukin 1 ◽  
Growth Control ◽  
Melanoma Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Functional Relevance ◽  
Antisense Oligomer ◽  
Negative Mutant

Induction of apoptosis by diverse exogenous signals is dependent on elevation of intracellular Ca2+. This process of cell death can be blocked by actinomycin D, indicating that it requires gene transcription events. To identify genes that are required for apoptosis, we used thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2+)-ATPase and thereby increases cytosolic Ca2+. Exposure to TG led to induction of the zinc finger transcription factor, EGR-1, and apoptosis in human melanoma cells, A375-C6. To determine the functional relevance of EGR-1 expression in TG-inducible apoptosis, we employed a dominant negative mutant which functionally competes with EGR-1 in these cells. Interestingly, the dominant negative mutant inhibited TG-inducible apoptosis. Consistent with this observation, an antisense oligomer directed against Egr-1 also led to a diminution of the number of cells that undergo TG-inducible apoptosis. These results suggest a novel regulatory role for EGR-1 in mediating apoptosis that is induced by intracellular Ca2+ elevation. We have previously shown that in these melanoma cells, EGR-1 acts to inhibit the growth arresting action of interleukin-1. Together, these results imply that EGR-1 plays inducer-specific roles in growth control.

scholarly journals The HTR1 Gene Is a Dominant Negative Mutant Allele of MTH1 and Blocks Snf3- and Rgt2-Dependent Glucose Signaling in Yeast

2000 ◽  
Vol 182 (2) ◽  
pp. 540-542 ◽  
Author(s):  
Frank Schulte ◽  
Roman Wieczorke ◽  
Cornelis P. Hollenberg ◽  
Eckhard Boles
Keyword(s):  
Gene Expression ◽  
Mutant Allele ◽  
Transcriptional Regulator ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hexose Transporter ◽  
Transporter Gene ◽  
Glucose Signaling ◽  
Mutant Forms ◽  
Negative Mutant

ABSTRACT Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose. We have cloned dominantHTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.

Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 901-909 ◽  
Author(s):  
E. Levine ◽  
C.H. Lee ◽  
C. Kintner ◽  
B.M. Gumbiner
Keyword(s):  
Early Embryo ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Truncated Form ◽  
Adhesive Properties ◽  
Negative Mutant ◽  
E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

scholarly journals Modulation of PKCδ signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3

2009 ◽  
Vol 296 (3) ◽  
pp. L519-L526 ◽  
Author(s):  
Neetu Sud ◽  
Sanjiv Kumar ◽  
Stephen Wedgwood ◽  
Stephen M. Black
Keyword(s):  
Nitric Oxide ◽  
Endothelial Nitric Oxide Synthase ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Enos Expression ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Negative Mutant

We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKCδ. In this study, we explore the role of PKCδ in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKCδ activation. Modulation of PKCδ before shear with a dominant negative mutant of PKCδ (DN PKCδ) or bryostatin (a known PKCδ activator) demonstrated that PKCδ inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKCδ activation inhibited these events. To gain insight into the mechanism by which PKCδ inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKCδ phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKCδ reduced, while PKCδ activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKCδ activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.

scholarly journals Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs

1999 ◽  
Vol 80 (8) ◽  
pp. 1965-1974 ◽  
Author(s):  
Anne Marie Szilvay ◽  
Stig-Ove Bøe ◽  
Karl-Henning Kalland
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Dominant Negative ◽  
Structural Proteins ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Negative Mutant

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the trans-dominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

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