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Author Gupta, M.L.J.; Carvalho, P.; Roof, D.M.; Pellman, D. file  url
openurl 
  Title Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle Type Journal Article
  Year 2006 Publication Nature Cell Biology Abbreviated Journal Nat Cell Biol  
  Volume 8 Issue 9 Pages 913-923  
  Keywords (up)  
  Abstract The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.

Subject headings: Adenosine Triphosphatases/metabolism; Cell Cycle/physiology; Kinesin/*metabolism; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology; Molecular Motor Proteins/*physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology; Tubulin/metabolism
 
  Call Number Serial 2212  
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Author Westermann, S.; Wang, H.-W.; Avila-Sakar, A.; Drubin, D.G.; Nogales, E.; Barnes, G. file  url
openurl 
  Title The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends Type Journal Article
  Year 2006 Publication Nature Abbreviated Journal Nature  
  Volume 440 Issue 7083 Pages 565-569  
  Keywords (up)  
  Abstract Chromosomes interact through their kinetochores with microtubule plus ends and they are segregated to the spindle poles as the kinetochore microtubules shorten during anaphase A of mitosis. The molecular natures and identities of coupling proteins that allow microtubule depolymerization to pull chromosomes to poles during anaphase have long remained elusive. In budding yeast, the ten-protein Dam1 complex is a critical microtubule-binding component of the kinetochore that oligomerizes into a 50-nm ring around a microtubule in vitro. Here we show, with the use of a real-time, two-colour fluorescence microscopy assay, that the ring complex moves processively for several micrometres at the ends of depolymerizing microtubules without detaching from the lattice. Electron microscopic analysis of 'end-on views' revealed a 16-fold symmetry of the kinetochore rings. This out-of-register arrangement with respect to the 13-fold microtubule symmetry is consistent with a sliding mechanism based on an electrostatically coupled ring-microtubule interface. The Dam1 ring complex is a molecular device that can translate the force generated by microtubule depolymerization into movement along the lattice to facilitate chromosome segregation.

Subject Headings: Cell Cycle Proteins/*physiology; Chromosome Segregation/physiology; Kinetochores/*physiology/ultrastructure; Microscopy, Fluorescence; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology/ultrastructure; Movement; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology/ultrastructure
 
  Call Number Serial 2219  
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Author Bieling, P.; Laan, L.; Schek, H.; Munteanu, E.L.; Sandblad, L.; Dogterom, M.; Brunner, D.; Surrey, T. file  url
openurl 
  Title Reconstitution of a microtubule plus-end tracking system in vitro Type Journal Article
  Year 2007 Publication Nature Abbreviated Journal Nature  
  Volume 450 Issue 7172 Pages 1100-1105  
  Keywords (up)  
  Abstract The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Subject Heading: Cell-Free System; Heat-Shock Proteins/metabolism; Intermediate Filament Proteins/metabolism; Microscopy, Fluorescence; Microtubule-Associated Proteins/*metabolism; Microtubules/*chemistry/*metabolism; *Schizosaccharomyces/chemistry/cytology; Schizosaccharomyces pombe Proteins/metabolism
 
  Call Number Serial 2223  
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Author Gupta, M.L.J.; Carvalho, P.; Roof, D.M.; Pellman, D. file  url
openurl 
  Title Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle Type Journal Article
  Year 2006 Publication Nature Cell Biology Abbreviated Journal Nat Cell Biol  
  Volume 8 Issue 9 Pages 913-923  
  Keywords (up)  
  Abstract The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.

Subject Headings: Adenosine Triphosphatases/metabolism; Cell Cycle/physiology; Kinesin/*metabolism; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology; Molecular Motor Proteins/*physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology; Tubulin/metabolism
 
  Call Number Serial 2260  
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Author Durieu-Trautmann, O.; Delavier-Klutchko, C. file  url
openurl 
  Title Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae Type Journal Article
  Year 1977 Publication Biochemical and Biophysical Research Communications Abbreviated Journal Biochem Biophys Res Commun  
  Volume 79 Issue 2 Pages 438-442  
  Keywords (up)  
  Abstract The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating and non sporulating cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.

Subject Headings: Ammonia/*pharmacology; DNA/biosynthesis; Fungal Proteins/biosynthesis; Glutamine/*pharmacology; Kinetics; RNA/biosynthesis; Saccharomyces cerevisiae/drug effects/*metabolism; Spores, Fungal/drug effects/metabolism
 
  Call Number Serial 2319  
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Author Favelukes, G.; Stoppani, A.O. url  openurl
  Title Baker's-yeast fumarase, a thiol enzyme Type Journal Article
  Year 1958 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 28 Issue 3 Pages 654-655  
  Keywords (up) *Hydro-Lyases; Saccharomyces cerevisiae/*metabolism; *Hydrases; *SACCHAROMYCES CEREVISIAE/metabolism  
  Abstract  
  Call Number Grinnell @ engelk @ Serial 483  
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Author Schmidt, B.H.; Osheroff, N.; Berger, J.M. file  url
openurl 
  Title Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity Type Journal Article
  Year 2012 Publication Nature Structural & Molecular Biology Abbreviated Journal Nat Struct Mol Biol  
  Volume 19 Issue 11 Pages 1147-1154  
  Keywords (up) Adenylyl Imidodiphosphate/*chemistry/metabolism; Amino Acid Sequence; Antigens, Neoplasm/*chemistry/metabolism; Chromatography, Gel; Crystallization; DNA/*chemistry/metabolism; DNA Topoisomerases, Type II/*chemistry/metabolism; DNA-Binding Proteins/*chemistry/metabolism; Dimerization; *Models, Molecular; Molecular Sequence Data; Multiprotein Complexes/*chemistry/metabolism; *Protein Conformation; Saccharomyces cerevisiae/*enzymology  
  Abstract Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.  
  Call Number Serial 2189  
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Author Van de Ven, W.J.; Creemers, J.W.; Roebroek, A.J. file  url
openurl 
  Title Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway Type Journal Article
  Year 1991 Publication Enzyme Abbreviated Journal Enzyme  
  Volume 45 Issue 5-6 Pages 257-270  
  Keywords (up) Animals; Binding Sites; Catalysis; Cloning, Molecular; Drosophila melanogaster; Furin; Humans; Invertebrate Hormones/genetics/metabolism; Mice; Models, Molecular; Multigene Family; Protein Conformation; Protein Precursors/*metabolism; *Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity; Subtilisins/genetics/*metabolism  
  Abstract Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.  
  Call Number Serial 524  
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Author Schenone, M.; Dancik, V.; Wagner, B.K.; Clemons, P.A. file  url
openurl 
  Title Target identification and mechanism of action in chemical biology and drug discovery Type Journal Article
  Year 2013 Publication Nature Chemical Biology Abbreviated Journal Nat Chem Biol  
  Volume 9 Issue 4 Pages 232-240  
  Keywords (up) Animals; Biomarkers, Pharmacological/chemistry/*metabolism; *Drug Discovery; *Drug Evaluation, Preclinical; *High-Throughput Screening Assays; Humans; Isotope Labeling; Mass Spectrometry; Molecular Targeted Therapy; Phenotype; RNA Interference; Reverse Genetics; Saccharomyces cerevisiae/drug effects/genetics/metabolism; Small Molecule Libraries/chemistry/*metabolism/pharmacology; Validation Studies as Topic  
  Abstract Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.  
  Call Number Serial 1592  
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Author Thacker, C.; Rose, A.M. file  url
doi  openurl
  Title A look at the Caenorhabditis elegans Kex2/Subtilisin-like proprotein convertase family Type Journal Article
  Year 2000 Publication BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology Abbreviated Journal Bioessays  
  Volume 22 Issue 6 Pages 545-553  
  Keywords (up) Animals; Caenorhabditis elegans/*enzymology/genetics; Genes, Helminth; Humans; Multigene Family; Mutation; Phylogeny; *Proprotein Convertases; *Saccharomyces cerevisiae Proteins; Subtilisins/chemistry/genetics/*metabolism  
  Abstract Significant advances have recently been made in our understanding of the mechanisms of activation of proteins that require processing. Often this involves endoproteolytic cleavage of precursor forms at basic residues, and is carried out by a group of serine endoproteinases, termed the proprotein convertases. In mammals, seven different convertases have been identified to date. These act in both the regulated secretory pathway for the processing of prohormones and proneuropeptides and in the constitutive secretory pathway, in which a variety of proproteins are activated endoproteolytically. The recently completed sequence of the nematode Caenorhabditis elegans genome affords a unique opportunity to examine the entire proprotein convertase family in a multicellular organism. Here we review the nature of the family, emphasising the structural features, characteristic of the four nematode genes, that supply all of the necessary functions unique to this group of serine endoproteinases. Studies of the C. elegans genes not only provide important information about the evaluation of this gene family but should help to illuminate the roles of these proteins in mammalian systems. BioEssays 22:545-553, 2000.  
  Call Number Serial 522  
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