The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a ␤-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.Self-propagating protein conformational changes have been proposed to be the cause of transmission of mammalian transmissible spongiform encephalopathies (1, 2), as well as for two yeast non-Mendelian inheritance elements, [PSI] and [URE3] (3-7). In the case of [PSI], an altered conformation of the Sup35 protein (Sup35p), which is the yeast homolog of the eukaryotic translation termination factor eRF3, is thought to be the determinant (3,8). Consistent with this proposal it was observed that the maintenance of [PSI] only requires the N-terminal 114-amino acid domain of Sup35p, and that overproduction of Sup35p or its N-terminal fragment in yeast induces the de novo appearance of [PSI] (9-11). Differential sedimentation and fluorescence microscopic studies further established a correlation between Sup35p coalescence and the appearance of [PSI], suggesting that ordered aggregation converts newly synthesized Sup35p into its like and is thus responsible for the propagation of the [PSI] element (12,13). In this study, we investigate the properties of the Sup35p polypeptide fragment consisting of residues 2-114, Sup35pN. It is found that Sup35pN aggregates to form amyloid-like filaments in vitro. We then show that seeding with preexisting filaments can speed up the formation of filaments from freshly prepared Sup35pN solutions. EXPERIMENTAL PROCEDURESProtein Purification and Filament Preparation. A DNA fragment encoding the N-terminal 114-residue segment of Sup35p, with an extra Met-Gly-Ser 2 -His 6 -Ser 2 -Gly 2 -Ser segment at the N terminus and a stop codon at the C terminus, was obtained by PCR from yeast genomic DNA, using appropriate oligonucleotides. The fragment was inserted into the expression vector pMW172 (14) and the protein was overexpressed in the BLR21(DE3)͞pLysS Escherichia coli strain (Novagen). Cell lysate was prepared in 20 mM Tris⅐HCl buffer (pH 7.9) containing 0.5 M NaCl, 5 mM imidazole, and 6 M guanidine hydrochloride (GdmCl), and loaded onto a Ni 2ϩ -NTA affinity column (Qiagen, Cha...

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

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Tittmann, P; Gross, H; Sonderegger, P. Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site. EMBO J. 1996EMBO J. , 15(9):2056. Postprint available at: http://www.zora.unizh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.unizh.ch Originally published at: EMBO J. 1996EMBO J. , 15(9):2056 Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site AbstractThe neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.The EMBO Journal vol.15 no.9 pp.2056-2068, 1996 Implications for the domain arrangement of axonin-1 derived from the mapping of

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.