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Mass spectrometry analysis of the kinetoplastid kinetochore protein KKT14 phosphorylated by the Aurora B kinase

ABSTRACT: We performed mass spectrometry of a recombinant KKT14 N-terminal fragment that was phosphorylated by AUK1 (Aurora B homolog in Trypanosoma brucei) in vitro to identify phosphorylation sites.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Bungo Akiyoshi

LAB HEAD: Bungo Akiyoshi

PROVIDER: PXD048677 | Pride | 2024-01-31

REPOSITORIES: Pride

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			Action	DRS
	KKT14NTR_by_AUK1_WT1.raw	Raw
	KKT14NTR_by_AUK1_WT2.raw	Raw
	KKT14NTR_by_AUK1_kinase_dead1.raw	Raw
	KKT14NTR_by_AUK1_kinase_dead2.raw	Raw
	Note_for_PRIDE_deposition_KKT14_kinase_assay.txt	Txt

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Mass spectrometry analysis of kinetochore proteins (KKT4, KKT14, and KKT22) in Trypanosoma brucei

Project description:We performed immunoprecipitation and mass spectrometry of kinetoplastid kinetochore proteins (full-length proteins for KKT4, KKT14, and KKT22 as well as KKT14 fragments) to identify co-purifying proteins.

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Mass spectrometry analysis of unconventional kinetoplastid kinetochore proteins KKT1, KKT2, and KKT6

Project description:The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. Here we performed chemical crosslinking mass spectrometry of the Trypanosoma brucei KKT2 DPB/KKT1C complex and KKT1/KKT6 complex. We also performed immunoprecipitation and mass spectrometry of KKT1 fragments (KKT1N and KKT1C) that were ectopically expressed and purified from trypanosomes.

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Mass spectrometry analysis of kinetoplastid kinetochore proteins (KKT23 acetyltransferase and its interactor KKT22) and histone acetylation levels in Trypanosoma brucei

Project description:KKT23 is a kinetoplastid kinetochore protein that has structural similarity to the GCN5 acetyltransferase domain. Using a kinetoplastid parasite Trypanosoma brucei, here we performed immunoprecipitation and mass spectrometry of GFP-tagged KKT23 N-terminal fragment (2–150) to identify co-purifying proteins, and immunoprecipitation and mass spectrometry of H2B-GFP after KKT23 depletion to examine acetylation states of histones. We also performed in vitro acetylation assays on recombinant trypanosome nucleosomes using recombinant KKT23 protein, followed by mass spectrometry.

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The kinetoplastid outer kinetochore

Project description:In spite of their universal function, the composition of kinetochores differs greatly across eukaryotes. Trypanosomes and other organisms Kinetoplastida diverged very early during eukaryotic radiation from the line that includes animals and fungi, and build kinetochores with very little detectable homology to models. However, the full extent of the trypanosome kinetochore is unclear. Here, we have used quantitative proteomics from multiple start points to define the interaction network of the kinetoplastid kinetochore-interacting proteins (KKIPs). These data identify new components of the kinetochore and define a stable 9-protein complex distal to KKIP1 that forms the majority of the kinetoplastid outer kinetochore.

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Trypanosome KKIP1 Dynamically Links the Inner Kinetochore to a Kinetoplastid Outer Kinetochore Complex.

Project description:Kinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of universal function, the composition of kinetochores in extant eukaryotes differs greatly. In trypanosomes and other Kinetoplastida, kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via an inner-kinetochore protein. However, we do not know the extent of the trypanosome kinetochore, and proteins interacting with a highly divergent Ndc80/Nuf2-like protein (KKIP1) suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. This complex incorporates proteins recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The outer kinetochore complex is physically distinct from inner-kinetochore proteins, but nanometer-scale label separation shows that KKIP1 bridges the two plates in the same orientation as Ndc80. Moreover, KKIP1 exhibits substantial elongation at metaphase, altering kinetochore structure in a manner consistent with pulling at the outer plate. Together, these data suggest that the KKIP1/KOK likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore, showing design parallels may exist in organisms with very different kinetochore composition.

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Divergent polo box domains underpin the unique kinetoplastid kinetochore.

Project description:Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1-19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here, we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei. KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region, and all three proteins have weak but significant similarity to the polo box domain (PBD) of PLK. These divergent PBDs of KKT2 and KKT20 are sufficient for kinetochore localization in vivo. We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids.

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Kinetoplastid kinetochore proteins KKT2 and KKT3 have unique centromere localization domains.

Project description: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.

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Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly.

Project description:Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.

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Identification of four unconventional kinetoplastid kinetochore proteins KKT22-25 in Trypanosoma brucei.

Project description: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.

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The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip-coupling protein.

Project description: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.

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