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Stefan Westermann

Publications and source records attributed to Stefan Westermann.

4 recordsLinked to original sources
PubMed2005

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex.

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.

Animals↗
PubMed2003

Architecture of the budding yeast kinetochore reveals a conserved molecular core.

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.

Amino Acid Sequence↗
PubMed2002

Identification of CfNek, a novel member of the NIMA family of cell cycle regulators, as a polypeptide copurifying with tubulin polyglutamylation activity in Crithidia.

Post-translational glutamylation of tubulin plays an important role in regulating the interaction between microtubules and associated proteins, but so far the enzymes involved in this process have not been cloned from any cellular source. Using a modified purification scheme that employs a hydroxyapaptite chromatography as the final step we identified a 54 kDa band as the major polypeptide copurifying with tubulin polyglutamylation activity from the trypanosomatid Crithidia fasciculata. Based on peptide sequence information we have cloned the corresponding cDNA and identify Crithidia p54 as a novel member (termed CfNek) of the NIMA family of putative cell cycle regulators. CfNek is a protein of 479 amino acids that contains an unusual protein kinase domain that lacks the glycine-rich loop in subdomain I. The protein also harbours a PEST sequence and a pleckstrin homology domain. The tubulin polyglutamylase preparation displays the beta-casein phosphorylation activity typical for NIMA related kinases. Recombinant His-tagged CfNek expressed in Crithidia localises to the flagellar attachment zone/basal body of the parasite. After purification on a Ni(2+)-column the recombinant enzyme preparation displays ATP-dependent tubulin polyglutamylation activity as well as casein-phosphorylation activity.

Amino Acid Sequence↗