scholarly journals Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

2004 ◽  
Vol 164 (5) ◽  
pp. 701-715 ◽  
Author(s):  
Matthias Versele ◽  
Jeremy Thorner
Keyword(s):  
Wild Type ◽  
Gtp Hydrolysis ◽  
Filament Assembly ◽  
Gtp Binding ◽  
Type Mutant ◽  
Binding Residues ◽  
Septin Filament ◽  
Assembly Defect

Assembly at the mother–bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

scholarly journals Functional insight into the role of Orc6 in septin complex filament formation in Drosophila

2015 ◽  
Vol 26 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Katarina Akhmetova ◽  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Origin Recognition Complex ◽  
Spatial Organization ◽  
Cell Cortex ◽  
Filament Formation ◽  
Gtp Binding ◽  
Complex Functions ◽  
Septin Filament

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

scholarly journals A Mutation Associated with CMT2A Neuropathy Causes Defects in Fzo1 GTP Hydrolysis, Ubiquitylation, and Protein Turnover

2009 ◽  
Vol 20 (23) ◽  
pp. 5026-5035 ◽  
Author(s):  
Elizabeth A. Amiott ◽  
Mickael M. Cohen ◽  
Yann Saint-Georges ◽  
Allan M. Weissman ◽  
Janet M. Shaw
Keyword(s):  
Axonal Degeneration ◽  
Mitochondrial Fusion ◽  
Peripheral Neuropathies ◽  
Wild Type ◽  
Gtpase Domain ◽  
Gtp Hydrolysis ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.

scholarly journals Intragenic suppression of a capsid assembly-defective P22 tailspike mutation.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 673-681 ◽  
Author(s):  
P A Maurides ◽  
J J Schwarz ◽  
P B Berget
Keyword(s):  
Amino Acid ◽  
Structural Defect ◽  
Salt Bridge ◽  
Mutant Form ◽  
Capsid Assembly ◽  
Wild Type ◽  
Tailspike Protein ◽  
Assembly Defect

Abstract The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

1991 ◽  
Vol 11 (1) ◽  
pp. 12-19 ◽  
Author(s):  
J Milner ◽  
E A Medcalf ◽  
A C Cook
Keyword(s):  
Tumor Progression ◽  
Carboxyl Terminus ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Dominant Effect ◽  
Type Mutant ◽  
Wild Type P53

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

scholarly journals Dictyostelium discoideum mutant synag 7 with altered G-protein-adenylate cyclase interaction

1988 ◽  
Vol 91 (2) ◽  
pp. 287-294
Author(s):  
B.E. Snaar-Jagalska ◽  
P.J. Van Haastert
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
High Speed ◽  
Wild Type ◽  
High Affinity ◽  
Gtp Binding

Previous results have shown that Dictyostelium discoideum mutant synag 7 is defective in the regulation of adenylate cyclase by receptor agonists in vivo and by GTP gamma S in vitro; the guanine nucleotide activation of adenylate cyclase is restored by the high-speed supernatant from wild-type cells. Here we report that in synag 7 membranes: (1) cyclic AMP receptors had normal levels and were regulated by guanine nucleotides as in wild-type; (2) GTP binding and high-affinity GTPase were reduced but still stimulated by cyclic AMP; (3) the supernatant from wild-type cells restored GTP binding to membranes of this mutant, and partly restored high-affinity GTPase activity; (4) the supernatant of synag 7 was ineffective in these reconstitutions and did not influence GTP binding and GTPase activities in mutant or wild-type membranes. These results suggest that the defect in mutant synag 7 is located between G-protein and adenylate cyclase, and not between receptor and G-protein. A factor in the supernatant is absent in synag 7 and appears to be essential for normal GTP binding, GTPase and activation of adenylate cyclase. This soluble heat-labile factor may represent a new molecule required for receptor- and G-protein-mediated activation of adenylate cyclase.

scholarly journals THE MOLECULAR BASIS OF DOMINANCE

Genetics ◽  
1981 ◽  
Vol 97 (3-4) ◽  
pp. 639-666 ◽  
Author(s):  
Henrik Kacser ◽  
James A Burns
Keyword(s):  
Catalytic Activity ◽  
Sensitivity Coefficient ◽  
Wild Type ◽  
Fractional Change ◽  
Systemic Property ◽  
Enzyme Change ◽  
Type Mutant

ABSTRACT The best known genes of microbes, mice and men are those that specify enzymes. Wild type, mutant and heterozygote for variants of such genes differ in the catalytic activity at the step in the enzyme network specified by the gene in question. The effect on the respective phenotypes of such changes in catalytic activity, however, is not defined by the enzyme change as estimated by in vitro determination of the activities obtained from the extracts of the three types. In vivo enzymes do not act in isolation, but are kinetically linked to other enzymes via their substrates and products. These interactions modify the effect of enzyme variation on the phenotype, depending on the nature and quantity of the other enzymes present. An output of such a system, say a flux, is therefore a systemic property, and its response to variation at one locus must be measured in the whole system. This response is best described by the sensitivity coefficient, Z, which is defined by the fractional change in flux over the fractional change in enzyme activity.

scholarly journals Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo.

1980 ◽  
Vol 86 (1) ◽  
pp. 330-334 ◽  
Author(s):  
M W Kirschner
Keyword(s):  
Actin Filaments ◽  
Spatial Organization ◽  
Complete Suppression ◽  
Gtp Hydrolysis ◽  
Spontaneous Polymerization ◽  
Filament Assembly ◽  
Selective Stabilization ◽  
The Stability

In this report, we examine how the cell can selectively stabilize anchored filaments and suppress spontaneous filament assembly. Because microtubules and actin filaments have an organized distribution in cells, the cell must have a mechanism for suppressing spontaneous and random polymerization. Though the mechanism for suppressing spontaneous polymerization is unknown, an unusual property of these filaments has been demonstrated recently, i.e., under steady-stae conditions, in vitro actin filaments and microtubules can exhibit a flux of subunits through the polymers called "treadmilling." In vivo, however, most, if not all, of these polymers are attached at one end to specific structures and treadmilling should not occur. The function of treadmilling in vivo is, therefore, unclear at present. However, as shown here, the same physicochemical property of coupling assembly to ATP or GTP hydrolysis that leads to treadmilling in vitro can act to selectively stabilize anchored polymers in vivo. I show here that the theory of treadmilling implies that the concentration of subunits necessary for assembly of the nonanchored polymer will in general be higher than the concentration necessary for the assembly of polymers anchored with a specific polarity. This disparity in the monomer concentrations required for assembly can lead to a selective stabilization of anchored polymers and complete suppression of spontaneous polymerization at apparent equilibrium in vivo. It is possible, therefore, that the phenomenon of treadmilling is an in vitro manifestation of a mechanism designed to use ATP or GTP hydrolysis to control the spatial organization of filaments in the cell.

scholarly journals Tumor suppressor p53: analysis of wild-type and mutant p53 complexes

1991 ◽  
Vol 11 (1) ◽  
pp. 12-19 ◽  
Author(s):  
J Milner ◽  
E A Medcalf ◽  
A C Cook
Keyword(s):  
Tumor Progression ◽  
Carboxyl Terminus ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Dominant Effect ◽  
Type Mutant ◽  
Wild Type P53

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

2021 ◽  
pp. 1-24
Author(s):  
Juho-Matti Renko ◽  
Arun Kumar Mahato ◽  
Tanel Visnapuu ◽  
Konsta Valkonen ◽  
Mati Karelson ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Dopamine Neurons ◽  
Dopamine Depletion ◽  
Wild Type ◽  
Disease Modifying Therapy ◽  
Immortalized Cells ◽  
Midbrain Dopamine ◽  
6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

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