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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):

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Bungo Akiyoshi

LAB HEAD: Bungo Akiyoshi

PROVIDER: PXD048677 | Pride | 2024-01-31

REPOSITORIES: Pride

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Dataset's files

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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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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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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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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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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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