scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

scholarly journals Structural analysis of the role of TPX2 in branching microtubule nucleation

2017 ◽  
Vol 216 (4) ◽  
pp. 983-997 ◽  
Author(s):  
Raymundo Alfaro-Aco ◽  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Structural Analysis ◽  
Xenopus Laevis ◽  
Mitotic Spindle ◽  
Microtubule Nucleation ◽  
Domain Organization ◽  
Ring Complex ◽  
The Right

The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2’s ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

scholarly journals In vitro fibrillogenesis of collagen II from pig vitreous humour

1995 ◽  
Vol 306 (3) ◽  
pp. 871-875 ◽  
Author(s):  
C Yang ◽  
H Notbohm ◽  
Y Açil ◽  
R Heifeng ◽  
S Bierbaum ◽  
...  
Keyword(s):  
Cross Sections ◽  
Soluble Fraction ◽  
Unit Length ◽  
Insoluble Fraction ◽  
Vitreous Humour ◽  
Collagen Types ◽  
Collagen Molecules ◽  
Collagen Ii

Collagen from pig vitreous humour was fractionated into a soluble and an insoluble fraction by centrifugation. Most of the collagen II in the soluble fraction was present as pN-collagen II (procollagen II without the C-terminal propeptide), besides smaller quantities of procollagen II, collagen II and two as yet unidentified alpha-chains of collagen II. Other collagen types may be present only in trace amounts. Collagen II of the insoluble fraction, which is mostly deposited in fibrillar aggregates, consists of both pN-collagen II and collagen II. To determine the possible role of collagen II precursors in the formation of the extracellular matrix of the vitreous humour these collagen molecules were purified and in vitro fibrillogenesis was used to demonstrate that pN-collagen II could form fibrils in mixtures with collagen II. These fibrils have a reduced mass per unit length depending on the content of pN-collagen in the mixture. Cross-sections of the newly formed fibrillar aggregates revealed a flattened shape. The incomplete processing of the precursors of collagen II may be part of regulatory mechanisms possibly controlling the formation of a translucent scaffold as is required in the vitreous humour.

scholarly journals Enzyme activity after resealing within ghost erythrocyte cells, and protection by α-crystallin against fructose-induced inactivation

2002 ◽  
Vol 368 (3) ◽  
pp. 865-874 ◽  
Author(s):  
Barry K. DERHAM ◽  
John J. HARDING
Keyword(s):  
Enzyme Activity ◽  
Molecular Chaperone ◽  
Soluble Fraction ◽  
Erythrocyte Ghosts ◽  
Ghost Cell ◽  
Chaperone Function ◽  
Zero Time ◽  
Soluble Fractions

The role of α-crystallin as a molecular chaperone has been shown in many in vitro studies. In the present paper, we report on the chaperone function of α-crystallin within resealed erythrocyte ghosts. Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24h. The ghost cell suspension was separated into soluble and membrane fractions. Five of the enzymes had significantly greater enzyme activity after 24h than the control within the soluble fractions. Fructation caused a decrease in enzyme activity (relative to the control). Resealing of α-crystallin within the ghost cell alongside the enzymes protected against inactivation by fructose within the soluble fraction.

scholarly journals Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.

1992 ◽  
Vol 119 (1) ◽  
pp. 17-25 ◽  
Author(s):  
N Ulitzur ◽  
A Harel ◽  
N Feinstein ◽  
Y Gruenbaum
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Membrane Vesicles ◽  
Embryo Cell ◽  
Drosophila Embryo ◽  
Sperm Chromatin ◽  
Naked Dna ◽  
Nuclear Envelope Assembly

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.

scholarly journals Role of the HSP70 Homologue from Chloroplasts in the Assembly of the Photosynthetic Apparatus

1993 ◽  
Author(s):  
Rachel Nechushtai ◽  
Parag Chitnis
Keyword(s):  
Protein Complexes ◽  
Photosynthetic Apparatus ◽  
Crop Productivity ◽  
Cdna Clones ◽  
Light Harvesting Complex ◽  
Complex Protein ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

The major goal of the proposed research was to study the role of a 70-kDa heat shock cognate protein from chloroplasts (ct-HSP70) in the assembly of chlorophyll-protein complexes. The latters are mostly important in allowing photosynthesis to occur. Photosynthesis is at the heart of crop productivity and the knowledge of the biogenesis of the photosynthetic apparatus is essential to manipulate the efficiency of photosynthesis. The characterization of the function of the ct-HSP70 was planned to be studied in vitro by assaying its capability to physically interact with the thylakoid proteins and to assist their assembly into thylakoid membranes. We planned to identify regions in the light-harvesting complex protein (LHCP) that interact with the ct-HSP70 and characterize the interaction between them. We also intended to isolate cDNA clones encoding ct-HSP70, sequence them, express one of them in E. coli and use the purified protein for functional assays. The research in this BARD proposal aimed at providing insights and aid in understanding the mechanism by which plants may respond to the heat stress. Since plants often experience increased temperatures.

scholarly journals Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Dolonchapa Chakraborty ◽  
Andrew J. Darwin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Protein Complexes ◽  
Associated Proteins ◽  
Carboxyl Terminal ◽  
Multiple Species ◽  
Processing Protease ◽  
Enzyme Complexes

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that assisting CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa . CtpA copurifies with the uncharacterized lipoprotein, LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis, and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the LbcA copurification partners are not cleaved by CtpA, but might copurify with LbcA because they participate in multi-enzyme complexes containing CtpA substrates. These findings are important, because CTPs and their associated proteins affect peptidoglycan remodeling and virulence in multiple species.

scholarly journals Granule regulation by phase separation during Drosophila oogenesis

2020 ◽  
Vol 4 (3) ◽  
pp. 355-364
Author(s):  
M. Sankaranarayanan ◽  
Timothy T. Weil
Keyword(s):  
Phase Separation ◽  
Translational Control ◽  
Protein Complexes ◽  
Physiological Role ◽  
Drosophila Oogenesis ◽  
And Function ◽  
Rna Protein Complexes ◽  
Liquid Liquid Phase Separation

Drosophila eggs are highly polarised cells that use RNA–protein complexes to regulate storage and translational control of maternal RNAs. Ribonucleoprotein granules are a class of biological condensates that form predominantly by intracellular phase separation. Despite extensive in vitro studies testing the physical principles regulating condensates, how phase separation translates to biological function remains largely unanswered. In this perspective, we discuss granules in Drosophila oogenesis as a model system for investigating the physiological role of phase separation. We review key maternal granules and their properties while highlighting ribonucleoprotein phase separation behaviours observed during development. Finally, we discuss how concepts and models from liquid–liquid phase separation could be used to test mechanisms underlying granule assembly, regulation and function in Drosophila oogenesis.

scholarly journals Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Dolonchapa Chakraborty ◽  
Andrew J Darwin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Type Iii Secretion ◽  
Protein Complexes ◽  
Peptidoglycan Hydrolases ◽  
Carboxyl Terminal ◽  
Processing Protease ◽  
Enzyme Complexes

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that facilitating CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates.

scholarly journals The Role of Xgrip210 in γ-Tubulin Ring Complex Assembly and Centrosome Recruitment

2000 ◽  
Vol 151 (7) ◽  
pp. 1525-1536 ◽  
Author(s):  
Lijun Zhang ◽  
Thomas J. Keating ◽  
Andrew Wilde ◽  
Gary G. Borisy ◽  
Yixian Zheng
Keyword(s):  
Immunogold Labeling ◽  
Structural Studies ◽  
Complex Assembly ◽  
Interacting Protein ◽  
Centrosomal Protein ◽  
Ring Complex

The γ-tubulin ring complex (γTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that γTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the γTuRC. Consequently, the cap structure of the γTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus γTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of γTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the γTuRC and that its localization is more distal, toward the tip of the γTuRC-cap structure, than that of γ-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the γTuRC, but also the recruitment of γ-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the γTuRC cap structure that is required for the assembly of the γTuRC.

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