scholarly journals Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.

1991 ◽  
Vol 112 (6) ◽  
pp. 1199-1203 ◽  
Author(s):  
M Schliwa ◽  
T Shimizu ◽  
R D Vale ◽  
U Euteneuer
Keyword(s):  
Cell Model ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Nucleoside Triphosphates ◽  
Membrane Bound ◽  
Atp Analogues ◽  
Nucleotide Specificity ◽  
Kinetics Of ◽  
Broad Specificity

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.

scholarly journals Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin.

1991 ◽  
Vol 112 (6) ◽  
pp. 1189-1197 ◽  
Author(s):  
T Shimizu ◽  
K Furusawa ◽  
S Ohashi ◽  
Y Y Toyoshima ◽  
M Okuno ◽  
...  
Keyword(s):  
Motor Proteins ◽  
Molecular Motor ◽  
Bovine Brain ◽  
Turnover Rates ◽  
Heavy Meromyosin ◽  
Substrate Specificities ◽  
Different Types ◽  
Atp Analogues ◽  
Nucleotide Specificity

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.

scholarly journals Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

1998 ◽  
Vol 143 (3) ◽  
pp. 589-599 ◽  
Author(s):  
Anne Spang ◽  
Randy Schekman
Keyword(s):  
Vesicle Trafficking ◽  
Retrograde Transport ◽  
Snare Proteins ◽  
Reporter Protein ◽  
In Vitro System ◽  
Retrograde Trafficking ◽  
Simple Set ◽  
Membrane Bound

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.

O n the Mechanism of Inactivation and ATP-Dependent Reactivation of Rat Liver Tyrosine Aminotransferase

1977 ◽  
Vol 32 (9-10) ◽  
pp. 777-780 ◽  
Author(s):  
Hans-Heinrich Hamm ◽  
Werner Seubert
Keyword(s):  
Rat Liver ◽  
Tyrosine Aminotransferase ◽  
Particulate Fraction ◽  
Kidney Cortex ◽  
Aminotransferase Activity ◽  
Liver And Kidney ◽  
Membrane Bound ◽  
Nucleotide Specificity

Abstract The mechanism of in vitro inactivation and ATP-dependent rapid reactivation of rat liver tyrosine aminotransferase by a membrane-bound system from rat liver and kidney cortex and the nucleotide specificity of this process was investigated using partially purified tyrosine amino­ transferase as a substrate. Adenosine 5′-triphosphate (ATP) could be replaced by guanosine 5′-tri-phosphate (GTP), whereas inosine 5′-triphosphate (ITP) was less effective. During reactivation [γ-32P]A T P was incorporated into the enzyme and not excorporated by incubation of the labeled enzyme with excess non-radioative ATP. Inactivation of labeled tyrosine aminotransferase by a particulate fraction led to a decrease protein-bound radioactivity concomitant with an increase of [32P] orthophosphate. This points to a phosphorylation and dephosphorylation mechanism in the regulation of tyrosine aminotransferase activity.

scholarly journals Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES) do not catalyse the refolding of mitochondrial malate dehydrogenase

1993 ◽  
Vol 291 (1) ◽  
pp. 139-144 ◽  
Author(s):  
A D Miller ◽  
K Maghlaoui ◽  
G Albanese ◽  
D A Kleinjan ◽  
C Smith
Keyword(s):  
Escherichia Coli ◽  
Kinetic Analysis ◽  
Malate Dehydrogenase ◽  
Molar Excess ◽  
Absolute Requirement ◽  
Nucleoside Triphosphates ◽  
Atp Analogues ◽  
Mitochondrial Malate Dehydrogenase ◽  
In Vitro Refolding

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.

scholarly journals Disease-associated mutations in human BICD2 hyperactivate motility of dynein–dynactin

2017 ◽  
Vol 216 (10) ◽  
pp. 3051-3060 ◽  
Author(s):  
Walter Huynh ◽  
Ronald D. Vale
Keyword(s):  
Spinal Muscular Atrophy ◽  
Hippocampal Neurons ◽  
Muscular Atrophy ◽  
Membrane Vesicle ◽  
Point Mutations ◽  
Neurite Growth ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Dominant Form

Bicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2’s membrane vesicle partner, the GTPase Rab6a. Rab6a–GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein–dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus versus plus end–directed microtubule motility in neurons may underlie spinal muscular atrophy.

scholarly journals Disease-Associated Mutations in Human BICD2 Hyperactivate Motility of Dynein-Dynactin

2017 ◽  
Author(s):  
Walter Huynh ◽  
Ronald D. Vale
Keyword(s):  
Spinal Muscular Atrophy ◽  
Hippocampal Neurons ◽  
Muscular Atrophy ◽  
Membrane Vesicle ◽  
Point Mutations ◽  
Neurite Growth ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Dominant Form

AbstractBicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2’s membrane vesicle partner, the GTPase Rab6a. Rab6a-GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein-dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus- versus plus-end-directed microtubule motility in neurons may underlie spinal muscular atrophy.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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