ABSTRACT
Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.
Subject(s)
Oocytes , Proteins , Pregnancy , Animals , Female , Oocytes/metabolism , Proteins/metabolism , Embryo, Mammalian/metabolism , Cytoskeleton , Ribosomes , Embryonic Development , MammalsABSTRACT
Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Crystallography, X-Ray , Kinetochores/chemistry , Multiprotein Complexes/chemistry , Animals , Aurora Kinase B/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins , Humans , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Models, Chemical , Multiprotein Complexes/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolismABSTRACT
Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Histones/metabolism , Kinetochores/metabolism , Mitosis , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , HeLa Cells , Histones/genetics , Humans , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Polo-Like Kinase 1ABSTRACT
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.
Subject(s)
Chromosomal Proteins, Non-Histone/ultrastructure , Kinetochores/physiology , Kinetochores/ultrastructure , Centromere/physiology , Centromere Protein A/metabolism , Centromere Protein A/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins , HeLa Cells , Humans , Kinetochores/metabolism , Microtubules/metabolism , Microtubules/physiology , Nuclear Proteins/metabolismABSTRACT
We study the formation of vesicle condensates induced by the protein synapsin, as a cell-free model system mimicking vesicle pool formation in the synapse. The system can be considered as an example of liquid-liquid phase separation (LLPS) in biomolecular fluids, where one phase is a complex fluid itself consisting of vesicles and a protein network. We address the pertinent question why the LLPS is self-limiting and stops at a certain size, i.e., why macroscopic phase separation is prevented. Using fluorescence light microscopy, we observe different morphologies of the condensates (aggregates) depending on the protein-to-lipid ratio. Cryogenic electron microscopy then allows us to resolve individual vesicle positions and shapes in a condensate and notably the size and geometry of adhesion zones between vesicles. We hypothesize that the membrane tension induced by already formed adhesion zones then in turn limits the capability of vesicles to bind additional vesicles, resulting in a finite condensate size. In a simple numerical toy model we show that this effect can be accounted for by redistribution of effective binding particles on the vesicle surface, accounting for the synapsin-induced adhesion zone.
ABSTRACT
Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.
Subject(s)
Kinetochores/chemistry , Kinetochores/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Autoantigens/metabolism , Centromere/chemistry , Centromere/genetics , Centromere/metabolism , Centromere Protein A , Chromosomal Proteins, Non-Histone/metabolism , Humans , Microtubules/metabolism , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism , Spindle ApparatusABSTRACT
Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.
Subject(s)
Kinetochores/chemistry , Microtubule-Associated Proteins/chemistry , Amino Acid Sequence , Centromere/chemistry , Chromosome Segregation , Crystallography, X-Ray , Escherichia coli/metabolism , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints , Microscopy, Electron , Microtubules/chemistry , Mitosis , Models, Molecular , Molecular Sequence Data , Plasmids/metabolism , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising â¼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Microfilament Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Humans , Microfilament Proteins/chemistry , Protein Binding , Protein Domains , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Protein Structure, Quaternary , Protein Transport , Substrate SpecificityABSTRACT
Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes and permits further assembly of all other kinetochore subunits in vitro with relative ratios closely matching those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module and may have important implications for transgenerational inheritance of centromeric chromatin.
Subject(s)
Histones , Kinetochores , Centromere/metabolism , Centromere Protein A/genetics , Centromere Protein A/metabolism , Humans , Kinetochores/metabolism , NucleosomesABSTRACT
Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.
Subject(s)
Electroporation , Molecular Imaging , Recombinant Proteins/metabolism , Cell Line , Chromosomes, Human/metabolism , Farnesyltranstransferase/metabolism , Green Fluorescent Proteins/metabolism , Humans , Hydrodynamics , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints , Mutation/genetics , PrenylationABSTRACT
Kinetochores are protein scaffolds coordinating the process of chromosome segregation in mitosis. Kinetochore components are organized in functionally and topologically distinct domains that are designed to connect the sister chromatids to the mitotic spindle. The inner kinetochore proteins are in direct contact with the centromeric DNA, whilst the outer kinetochore proteins are responsible for binding to spindle microtubules. The conserved Ndc80 complex is implicated in several essential outer kinetochore functions, including microtubule binding and control of a safety device known as the spindle assembly checkpoint. Here, we describe how current work is contributing to unravel the complex endeavors of this essential kinetochore complex.
Subject(s)
Kinetochores/chemistry , Kinetochores/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Humans , Microtubules/metabolism , Models, Biological , Models, Molecular , Multiprotein Complexes , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/metabolismABSTRACT
Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18ß subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18ß 4:2 hexamer binds two copies of M18BP1 through M18BP1's 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18ß, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18ß scaffold.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CDC2 Protein Kinase/metabolism , Centromere Protein A/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Protein Multimerization , Cell Cycle Proteins , Centromere/metabolism , Humans , Phosphorylation , Protein Binding , Protein Processing, Post-TranslationalABSTRACT
Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.
Subject(s)
Centromere Protein A/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Cell Line , Centromere/chemistry , Centromere Protein A/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , DNA/chemistry , Humans , Kinetochores/chemistry , Kinetochores/metabolism , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αß'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.
Subject(s)
Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/physiology , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Dyneins/metabolism , HeLa Cells , Humans , Kinetochores/physiology , M Phase Cell Cycle Checkpoints/physiology , Microtubules/metabolism , Mitosis/physiology , Protein Transport/physiologyABSTRACT
Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chlorobium/enzymology , Guanosine Triphosphate/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Parkinson Disease/enzymology , Bacterial Proteins/genetics , Chlorobium/chemistry , Chlorobium/genetics , Dimerization , Humans , Hydrolysis , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Mutation , Parkinson Disease/genetics , Phosphorylation , Protein Structure, TertiaryABSTRACT
Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.
Subject(s)
Drosophila melanogaster/metabolism , Kinetochores/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Drosophila melanogaster/genetics , Humans , Kinetochores/chemistry , Kinetochores/ultrastructure , Mass Spectrometry , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins , Sequence AlignmentABSTRACT
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore-microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.
Subject(s)
Cell Cycle Proteins/chemistry , Kinetochores/metabolism , Microtubule-Associated Proteins/chemistry , Microtubules/metabolism , Mitosis , Spindle Apparatus/metabolism , Amino Acid Sequence , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosome Segregation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HeLa Cells , Humans , Kinetochores/ultrastructure , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Spindle Apparatus/ultrastructure , Tubulin/chemistry , Tubulin/genetics , Tubulin/metabolismABSTRACT
Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Protein Multimerization , CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Cytoskeletal Proteins , Macromolecular Substances/ultrastructure , Microscopy, Electron , Phosphorylation , Protein Processing, Post-TranslationalABSTRACT
H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.
Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Models, Molecular , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon Isotopes , DNA, Bacterial/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Glucose/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/chemistry , Proline/metabolism , Protein Conformation , Protein Folding , Protein Structure, Secondary , Salmonella typhimurium/chemistry , Sequence Deletion , Solutions , Structure-Activity Relationship , Water/chemistryABSTRACT
The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.