Targeting of GLUT6 (formerly GLUT9) and GLUT8 in rat adipose cells

2001 ◽  
Vol 358 (2) ◽  
pp. 517-522 ◽  
Author(s):  
Ivonne LISINSKI ◽  
Annette SCHÜRMANN ◽  
Hans-Georg JOOST ◽  
Samuel W. CUSHMAN ◽  
Hadi AL-HASANI
Keyword(s):  
Plasma Membrane ◽  
Constitutive Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Wild Type ◽  
Subcellular Targeting ◽  
Intracellular Compartments ◽  
Adipose Cells ◽  
Negative Mutant

The subcellular targeting of the two recently cloned novel mammalian glucose transporters, GLUT6 {previously referred to as GLUT9 [Doege, Bocianski, Joost and Schürmann (2000) Biochem. J. 350, 771–776]} and GLUT8, was analysed by expression of haemagglutinin (HA)-epitope-tagged GLUTs in transiently transfected primary rat adipose cells. Similar to HA-GLUT4, both transporters, HA-GLUT6 and HA-GLUT8, were retained in intracellular compartments in non-stimulated cells. In contrast, mutation of the N-terminal dileucine motifs in both constructs led to constitutive expression of the proteins on the plasma membrane. Likewise, when endocytosis was blocked by co-expression of a dominant-negative mutant of the dynamin GTPase, wild-type HA-GLUT6 and HA-GLUT8 accumulated on the cell surface. However, in contrast with HA-GLUT4, no translocation of HA-GLUT6 and HA-GLUT8 to the plasma membrane was observed when the cells were stimulated with insulin, phorbol ester or hyperosmolarity. Thus GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells, but are unresponsive to stimuli that induce translocation of GLUT4.

An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures

2000 ◽  
Vol 78 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Claudio Akio Masuda ◽  
Mónica Montero-Lomelí
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Internal Peptide ◽  
Limited Trypsinolysis ◽  
Wild Type Yeast ◽  
Negative Mutant

The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant 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 c-Cbl Mediates Ubiquitination, Degradation, and Down-regulation of Human Protease-activated Receptor 2

2005 ◽  
Vol 280 (16) ◽  
pp. 16076-16087 ◽  
Author(s):  
Claire Jacob ◽  
Graeme S. Cottrell ◽  
Daphne Gehringer ◽  
Fabien Schmidlin ◽  
Eileen F. Grady ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ring Finger ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Wild Type ◽  
Lysosomal Degradation ◽  
Proteolytic Activation ◽  
Gpcr Signaling ◽  
Early Endosomes ◽  
Protease Activated Receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2to irrevocably terminate signaling by this and perhaps other GPCRs.

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 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 Rab22a Regulates the Recycling of Membrane Proteins Internalized Independently of Clathrin

2004 ◽  
Vol 15 (8) ◽  
pp. 3758-3770 ◽  
Author(s):  
Roberto Weigert ◽  
Albert Chi Yeung ◽  
Jean Li ◽  
Julie G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Tubular Structures ◽  
Tubule Formation ◽  
Early Endosomes ◽  
Interfering Rna ◽  
Negative Mutant

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.

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