RESUMEN
Kinetochores form the interface between chromosomes and spindle microtubules and are thus under tight control by a complex regulatory circuitry. The Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint. Intriguingly, Aurora B is conserved even in kinetoplastids, a group of early-branching eukaryotes which possess a unique set of kinetochore proteins. It remains unclear how their kinetochores are regulated to ensure faithful chromosome segregation. Here, we show in Trypanosoma brucei that Aurora B activity controls the metaphase-to-anaphase transition through phosphorylation of the divergent Bub1-like protein KKT14. Depletion of KKT14 overrides the metaphase arrest resulting from Aurora B inhibition, while expression of non-phosphorylatable KKT14 delays anaphase onset. Finally, we demonstrate that re-targeting Aurora B to the outer kinetochore suffices to promote mitotic exit but causes extensive chromosome missegregation in anaphase. Our results indicate that Aurora B and KKT14 are involved in an unconventional circuitry controlling cell cycle progression in trypanosomes.
Asunto(s)
Anafase , Aurora Quinasa B , Segregación Cromosómica , Cinetocoros , Proteínas Protozoarias , Trypanosoma brucei brucei , Aurora Quinasa B/metabolismo , Aurora Quinasa B/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/enzimología , Cinetocoros/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Fosforilación , Huso Acromático/metabolismo , Huso Acromático/genética , Microtúbulos/metabolismo , Microtúbulos/genéticaRESUMEN
Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.
Asunto(s)
Cinetocoros , Proteínas Protozoarias , Cinetocoros/metabolismo , Cinetocoros/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Modelos Moleculares , Secuencia de Aminoácidos , Filogenia , Unión Proteica , Cristalografía por Rayos X , Segregación Cromosómica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genéticaRESUMEN
The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.
Asunto(s)
Cinesinas , Trypanosoma , Humanos , Survivin , Citoesqueleto , MitosisRESUMEN
KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463-645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.
Asunto(s)
Cinetocoros , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Trypanosoma brucei brucei , Cinetocoros/metabolismo , Cinetocoros/química , Secuencia de Aminoácidos , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins in vitro, including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.
RESUMEN
Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei, an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in T. brucei. As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of deGradFP in a cell line that had both alleles of KKT3 tagged with yellow fluorescent protein (YFP) caused a more severe growth defect than RNAi in procyclic (insect form) cells. deGradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the ease in making GFP fusion cell lines in T. brucei, deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates.
RESUMEN
Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Cinetocoros/metabolismo , Proteínas Protozoarias/metabolismo , Segregación Cromosómica , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismoRESUMEN
Variant surface glycoprotein (VSG) coats bloodstream form Trypanosoma brucei parasites, and monoallelic VSG expression underpins the antigenic variation necessary for pathogenicity. One of thousands of VSG genes is transcribed by RNA polymerase I in a singular nuclear structure called the expression site body (ESB), but how monoallelic VSG transcription is achieved remains unclear. Using a localization screen of 153 proteins we found one, ESB-specific protein 1 (ESB1), that localized only to the ESB and is expressed only in VSG-expressing life cycle stages. ESB1 associates with DNA near the active VSG promoter and is necessary for VSG expression, with overexpression activating inactive VSG promoters. Mechanistically, ESB1 is necessary for recruitment of a subset of ESB components, including RNA polymerase I, revealing that the ESB has separately assembled subdomains. Because many trypanosomatid parasites have divergent ESB1 orthologues yet do not undergo antigenic variation, ESB1 probably represents an important class of transcription regulators.
Asunto(s)
Trypanosoma brucei brucei , Variación Antigénica/genética , Glicoproteínas de Membrana/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Factores de Transcripción/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Kinetochores are the macromolecular protein complexes that govern chromosome movement by binding spindle microtubules during mitosis and meiosis. Centromeres are the specific chromosomal regions that serve as the platform on which kinetochores assemble. Despite their essentiality for proper chromosome segregation, the size and organization of centromeres vary dramatically between species, while different compositions of kinetochores are found among eukaryotes. Here we discuss recent progress in understanding centromeres and kinetochores in non-traditional model eukaryotes. We specifically focus on select lineages (holocentric insects, early diverging fungi, and kinetoplastids) that lack CENP-A, a centromere-specific histone H3 variant that is critical for kinetochore specification and assembly in many eukaryotes. We also highlight some organisms that might have hitherto unknown types of kinetochore proteins.
Asunto(s)
Proteínas Cromosómicas no Histona , Cinetocoros , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , MitosisRESUMEN
The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
Asunto(s)
Cinetocoros/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Relación Estructura-Actividad , Trypanosoma brucei brucei/genética , Zinc/metabolismoRESUMEN
Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP2-3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.
Asunto(s)
Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas Protozoarias/metabolismo , Complejo Sinaptonémico/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas Protozoarias/genética , Complejo Sinaptonémico/genética , Trypanosoma brucei brucei/genéticaRESUMEN
The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.
Asunto(s)
Cinetocoros/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Protozoarias/química , Sitios de Unión , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Unión Proteica , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/metabolismoRESUMEN
KKT4 is a kinetoplastid-specific microtubule-binding kinetochore protein that lacks significant similarity to any known kinetochore or microtubule-binding proteins. Here we present the 1H, 13C and 15N resonance assignments for several fragments from the microtubule-binding domain of KKT4 (KKT4115-343) from Trypanosoma brucei. These assignments provide the starting point for detailed investigations of the structure, dynamics and interactions of the microtubule-binding region of KKT4.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Proteínas Protozoarias/análisis , Proteínas Protozoarias/química , Trypanosoma brucei brucei/metabolismo , Isótopos de Nitrógeno , Dominios ProteicosRESUMEN
The kinetochore is a macromolecular protein complex that drives chromosome segregation in eukaryotes. Unlike most eukaryotes that have canonical kinetochore proteins, evolutionarily divergent kinetoplastids, such as Trypanosoma brucei, have unconventional kinetochore proteins. T. brucei also lacks a canonical spindle checkpoint system, and it therefore remains unknown how mitotic progression is regulated in this organism. Here, we characterized, in the procyclic form of T. brucei, two paralogous kinetochore proteins with a CLK-like kinase domain, KKT10 and KKT19, which localize at kinetochores in metaphase but disappear at the onset of anaphase. We found that these proteins are functionally redundant. Double knockdown of KKT10 and KKT19 led to a significant delay in the metaphase to anaphase transition. We also found that phosphorylation of two kinetochore proteins, KKT4 and KKT7, depended on KKT10 and KKT19 in vivo Finally, we showed that the N-terminal part of KKT7 directly interacts with KKT10 and that kinetochore localization of KKT10 depends not only on KKT7 but also on the KKT8 complex. Our results reveal that kinetochore localization of KKT10 and KKT19 is tightly controlled to regulate the metaphase to anaphase transition in T. bruceiThis article has an associated First Person interview with the first author of the paper.
Asunto(s)
Trypanosoma brucei brucei , Proteínas de Ciclo Celular/genética , Segregación Cromosómica/genética , Humanos , Cinetocoros , Metafase , Proteínas Protozoarias/genética , Huso Acromático , Trypanosoma brucei brucei/genéticaRESUMEN
The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1-20). In addition, 12 proteins (KKT-interacting proteins 1-12, KKIP1-12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22-25, in Trypanosoma brucei. KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N-acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.
Asunto(s)
Cinetocoros/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Inmunoprecipitación , Proteínas Protozoarias/aislamiento & purificaciónRESUMEN
Centromeres in eukaryotes can be classified into three categories: point centromeres, regional centromeres, or holocentric. Now, a hybrid-type centromere is found in a pathogenic fungus that lacks the key kinetochore component CENP-A.
Asunto(s)
Histonas/genética , Mucor , Centrómero , Proteína A Centromérica , Proteínas Cromosómicas no Histona , CinetocorosRESUMEN
An Amendment to this Article has been published and is linked from the HTML version of this paper.
RESUMEN
Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.
Asunto(s)
Segregación Cromosómica , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Microtúbulos/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Kinetoplastids have a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. These single-copy organelles must be duplicated and segregated faithfully to daughter cells at each cell division. In Trypanosoma brucei, although duplication of both organelles starts around the same time, segregation of the kinetoplast precedes that of the nucleus. Cytokinesis subsequently takes place so that daughter cells inherit a single copy of each organelle. Very little is known about the molecular mechanism that governs the timing of these events. Furthermore, it is thought that T. brucei lacks a spindle checkpoint that delays the onset of nuclear division in response to spindle defects. Here we show that a mitotic cyclin CYC6 has a dynamic localization pattern during the cell cycle, including kinetochore localization. Using CYC6 as a molecular cell cycle marker, we confirmed that T. brucei cannot delay the onset of anaphase in response to a bipolar spindle assembly defect. Interestingly, expression of a stabilized form of CYC6 caused the nucleus to arrest in a metaphase-like state without preventing cytokinesis. We propose that trypanosomes have an ability to regulate the timing of nuclear division by modulating the CYC6 protein level, without a spindle checkpoint.