ABSTRACT
Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3-Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module.
Subject(s)
Chromosome Segregation , Microtubule-Associated Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Amino Acids/metabolism , Animals , Binding Sites , Crystallography, X-Ray , Microtubule-Associated Proteins/chemistry , Microtubules/metabolism , Models, Molecular , Protein Binding , Protein Domains , Schizosaccharomyces pombe Proteins/chemistryABSTRACT
Correct segregation of duplicated chromosomes to daughter cells during mitosis requires the action of the cohesin complex. This tripartite ring-shaped molecule is involved in holding replicated sister chromatids together from S phase until anaphase onset. Establishment of stable cohesion involves acetylation of the Smc3 component of cohesin during replication by the Eco1 acetyltransferase. This has been proposed to antagonise the activity of another member of the cohesin complex, Wpl1. Here, we describe the X-ray structure of the conserved Wapl domain, and demonstrate that it binds the ATPase head of the Smc3 protein. We present data that suggest that Wpl1 may be involved in regulating the ATPase activity of cohesin, and that this may be subject to the acetylation state of Smc3. In addition, we present a structure of the Wapl domain bound to a functionally relevant segment of the Smc3 ATPase.
Subject(s)
Ascomycota/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Fungal Proteins/chemistry , Gene Expression Regulation, Fungal , Acetylation , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Catalytic Domain , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Crystallography, X-Ray , DNA Replication , Fluorescence Polarization , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptide Fragments/metabolism , Protein Array Analysis , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , CohesinsABSTRACT
A novel class of molecular chaperones co-ordinates the assembly and targeting of complex metalloproteins by binding to an amino-terminal peptide of the cognate substrate. We have previously shown that the NarJ chaperone interacts with the N-terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study, NMR structural analysis revealed that the NarG(1-15) peptide adopts an alpha-helical conformation in solution. Moreover, NarJ recognizes and binds the helical NarG(1-15) peptide mostly via hydrophobic interactions as deduced from isothermal titration calorimetry analysis. NMR and differential scanning calorimetry analysis revealed a modification of NarJ conformation during complex formation with the NarG(1-15) peptide. Isothermal titration calorimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction.
