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 ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Negative Mutant

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 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.

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.

scholarly journals Cytoplasmic linker proteins promote microtubule rescue in vivo

2002 ◽  
Vol 159 (4) ◽  
pp. 589-599 ◽  
Author(s):  
Yulia A. Komarova ◽  
Anna S. Akhmanova ◽  
Shin-ichiro Kojima ◽  
Niels Galjart ◽  
Gary G. Borisy
Keyword(s):  
Plasma Membrane ◽  
Cho Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Asymmetric Behavior ◽  
Head Domain ◽  
Negative Mutant

The role of plus end–tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.

scholarly journals A Dominant-negative Mutant of Androgen Receptor Coregulator ARA54 Inhibits Androgen Receptor-mediated Prostate Cancer Growth

2001 ◽  
Vol 277 (7) ◽  
pp. 4609-4617 ◽  
Author(s):  
Hiroshi Miyamoto ◽  
Mujib Rahman ◽  
Hiroshi Takatera ◽  
Hong-Yo Kang ◽  
Shuyuan Yeh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Dominant Negative ◽  
Cancer Growth ◽  
Dominant Negative Mutant ◽  
Negative Mutant

scholarly journals UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53

2002 ◽  
Vol 365 (1) ◽  
pp. 133-145 ◽  
Author(s):  
Nadine CHOUINARD ◽  
Kristoffer VALERIE ◽  
Mahmoud ROUABHIA ◽  
Jacques HUOT
Keyword(s):  
Adaptive Response ◽  
Human Keratinocytes ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Normal Human Keratinocytes ◽  
Mitogen Activated Protein ◽  
Normal Human ◽  
Induced Apoptosis ◽  
Negative Mutant

Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

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