scholarly journals The Structure of the γ-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation

2008 ◽  
Vol 19 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Justin M. Kollman ◽  
Alex Zelter ◽  
Eric G.D. Muller ◽  
Bethany Fox ◽  
Luke M. Rice ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Higher Order ◽  
The Other ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Evolutionarily Conserved ◽  
Microtubule Growth ◽  
Order Complexes

The γ-tubulin small complex (γ-TuSC) is an evolutionarily conserved heterotetramer essential for microtubule nucleation. We have determined the structure of the Saccharomyces cerevisiae γ-TuSC at 25-Å resolution by electron microscopy. γ-TuSC is Y-shaped, with an elongated body connected to two arms. Gold labeling showed that the two γ-tubulins are located in lobes at the ends of the arms, and the relative orientations of the other γ-TuSC components were determined by in vivo FRET. The structures of different subpopulations of γ-TuSC indicate flexibility in the connection between a mobile arm and the rest of the complex, resulting in variation of the relative positions and orientations of the γ-tubulins. In all of the structures, the γ-tubulins are distinctly separated, a configuration incompatible with the microtubule lattice. The separation of the γ-tubulins in isolated γ-TuSC likely plays a role in suppressing its intrinsic microtubule-nucleating activity, which is relatively weak until the γ-TuSC is incorporated into higher order complexes or localized to microtubule-organizing centers. We propose that further movement of the mobile arm is required to bring the γ-tubulins together in microtubule-like interactions, and provide a template for microtubule growth.

scholarly journals Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex.

1995 ◽  
Vol 15 (7) ◽  
pp. 3487-3495 ◽  
Author(s):  
M P Draper ◽  
C Salvadore ◽  
C L Denis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Significant Similarity ◽  
Eukaryotic Transcription ◽  
C Elegans ◽  
Mouse Protein ◽  
Transcriptional Regulatory ◽  
Evolutionarily Conserved ◽  
Regulatory Complex

The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

scholarly journals Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

2019 ◽  
Vol 30 (12) ◽  
pp. 1555-1574 ◽  
Author(s):  
Maria Nieves Martinez Marshall ◽  
Anita Emmerstorfer-Augustin ◽  
Kristin L. Leskoske ◽  
Lydia H. Zhang ◽  
Biyun Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Lipid Metabolism ◽  
Eukaryotic Cell ◽  
Target Of Rapamycin ◽  
Multiprotein Complex ◽  
Ph Domain ◽  
Evolutionarily Conserved ◽  
And Function

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

scholarly journals Alp7-Mto1 and Alp14 synergize to promote interphase microtubule regrowth from the nuclear envelope

2019 ◽  
Vol 11 (11) ◽  
pp. 944-955 ◽  
Author(s):  
Wenyue Liu ◽  
Fan Zheng ◽  
Yucai Wang ◽  
Chuanhai Fu
Keyword(s):  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Microtubule Assembly ◽  
Dependent Manner ◽  
Microtubule Nucleation ◽  
Microtubule Organizing Centers ◽  
Evolutionarily Conserved ◽  
Microtubule Growth ◽  
In Cells

Abstract Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE.

On The Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex

1998 ◽  
Vol 4 (S2) ◽  
pp. 954-955
Author(s):  
James K. Stoops ◽  
Z. Hong Zhou ◽  
John P. Schroeter ◽  
Steven J. Kolodziej ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Structural Organization ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Oxidative Decarboxylation ◽  
Multienzyme Complex ◽  
The Core ◽  
Dehydrogenase Complex

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

scholarly journals Effect of denture surface glazing on denture plaque formation

2005 ◽  
Vol 16 (2) ◽  
pp. 129-134 ◽  
Author(s):  
Newton Sesma ◽  
Dalva Cruz Laganá ◽  
Susana Morimoto ◽  
Carlos Gil
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Bacterial Colonization ◽  
Plaque Formation ◽  
The Other ◽  
Bacterial Plaque ◽  
The Right ◽  
Clinical Experiment ◽  
Scanning Electron

This study evaluated, in vivo, the efficacy of a denture glazing material (Palaseal) in modifying plaque colonization of dentures. Ten subjects were selected and received maxillary temporary partial removable dentures, with complete acrylic palatal coverage. The right half of the fitting surface of the denture bases were glazed with Palaseal, whereas the other half was not glazed. One month after insertion, two fragments of the resin base of all dentures were removed (one from the glazed side and another from the non-glazed side). These samples were prepared and examined by scanning electron microscopy. Three months after insertion, other fragments were obtained and analyzed. Microscopic observation at 1 month revealed that, for all patients, the plaque film was thinner on the treated side in comparison to the non-treated side. However, at the 3-month evaluation, some areas of the glaze showed cracking, and both glazed and non-glazed sides were covered by a dense bacterial plaque film. In conclusion, the findings of this clinical experiment showed that glazing denture's fitting surface did not prevent bacterial colonization, but favored plaque removal while the glaze layer remained intact. After three months, glaze cracks created microretentive areas that increased plaque accumulation.

scholarly journals 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae

2017 ◽  
Vol 114 (33) ◽  
pp. 8782-8787 ◽  
Author(s):  
Jing Huang ◽  
Zhouqing Luo ◽  
Wantao Ying ◽  
Qichen Cao ◽  
He Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Set ◽  
Histone Lysine ◽  
Lysine Deacetylase ◽  
Chronological Life Span ◽  
Evolutionarily Conserved ◽  
A Cell ◽  
Low Glucose ◽  
Carbon Transport

New types of modifications of histones keep emerging. Recently, histone H4K8 2-hydroxyisobutyrylation (H4K8hib) was identified as an evolutionarily conserved modification. However, how this modification is regulated within a cell is still elusive, and the enzymes adding and removing 2-hydroxyisobutyrylation have not been found. Here, we report that the amount of H4K8hib fluctuates in response to the availability of carbon source in Saccharomyces cerevisiae and that low-glucose conditions lead to diminished modification. The removal of the 2-hydroxyisobutyryl group from H4K8 is mediated by the histone lysine deacetylase Rpd3p and Hos3p in vivo. In addition, eliminating modifications at this site by alanine substitution alters transcription in carbon transport/metabolism genes and results in a reduced chronological life span (CLS). Furthermore, consistent with the glucose-responsive H4K8hib regulation, proteomic analysis revealed that a large set of proteins involved in glycolysis/gluconeogenesis are modified by lysine 2-hydroxyisobutyrylation. Cumulatively, these results established a functional and regulatory network among Khib, glucose metabolism, and CLS.

Dimerization of presenilin-1 in vivo: suggestion of novel regulatory mechanisms leading to higher order complexes

2003 ◽  
Vol 301 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Sébastien S Hébert ◽  
Chantal Godin ◽  
Takami Tomiyama ◽  
Hiroshi Mori ◽  
Georges Lévesque
Keyword(s):  
Higher Order ◽  
Regulatory Mechanisms ◽  
Presenilin 1 ◽  
Order Complexes

scholarly journals Yeast Bax Inhibitor (Bxi1p/Ybh3p) is a Calcium Channel in E. coli

2019 ◽  
Author(s):  
James Mullin ◽  
John Kalhorn ◽  
Nicholas Mello ◽  
Amanda Raffa ◽  
Alexander Strakosha ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Calcium Channel ◽  
The Other ◽  
Bacterial Cells ◽  
E Coli ◽  
Founding Member ◽  
Bona Fide ◽  
Small Molecular Inhibitors

AbstractHuman Bax Inhibitor-1 (HsBI-1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor-containing motif (TMBIM). Mechanistically, BI-1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH-sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function of Bxi1p/Ybh3p, we overexpressed Bxi1p-EGFP in E. coli to determine if it is a calcium channel. We show that bacterial cells expressing Bxi1p-EGFP are more permeable to calcium than controls. Thus, our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. Further, we use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small-molecular inhibitors of this clinically-important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor-1 (HsBI-1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium.

scholarly journals Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

2018 ◽  
Author(s):  
Andrew Lyon ◽  
Alex Zelter ◽  
Shruthi Viswanath ◽  
Alison Maxwell ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
The Other ◽  
Assembly Process ◽  
X Ray ◽  
X Ray Crystallography ◽  
Small Complex ◽  
Ring Complex ◽  
Biochemical Analyses

AbstractMicrotubule (MT) nucleation in vivo is regulated by the γ-tubulin ring complex (γTuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast γTuRC is composed of seven γ-tubulin small complex (γTuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple γTuSCs, thereby stabilizing the otherwise low-affinity interface between γTuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with γTuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the γTuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and γTuSC and the other stabilizing the interface between adjacent γTuSCs.

Tissue and Hepatic Subcellular Distribution of Liposomes Containing Bleomycin after Intravenous Administration to Patients with Neoplasms

1976 ◽  
Vol 51 (4) ◽  
pp. 421-425 ◽  
Author(s):  
A. W. Segal ◽  
G. Gregoriadis ◽  
J. P. Lavender ◽  
D. Tarin ◽  
T. J. Peters
Keyword(s):  
Electron Microscopy ◽  
Tissue Distribution ◽  
Subcellular Distribution ◽  
The Other ◽  
Whole Body ◽  
Post Mortem ◽  
Malignant Tissue ◽  
Body Scanning ◽  
Liver Biopsies

1. Liposomes containing 111In-labelled bleomycin were injected intravenously into two patients. One patient had a hepatoma and the other had secondary adenocarcinomatous deposits in the liver. 2. The tissue distribution of 111In was determined by whole-body scanning and by measurement of the radioactivity in organs at autopsy. 3. Scans in vivo and post-mortem measurement of radioactivity indicated that liposomes accumulate predominantly in the liver, but that there is no selective uptake of liposomes by the malignant tissue. 4. The subcellular distribution of radioactivity in the liver was measured 90 min after injection by fractionation of percutaneous liver biopsies on sucrose density gradients. 5. Radioactivity within the liver was concentrated in lysosomes. 6. Electron microscopy of tissue obtained before the administration of liposomes revealed particles morphologically indistinguishable from liposomes in hepatoma cells and hepatocytes.

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