The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics

Abstract

SummaryDespite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.

Ki67 Antigen Contributes to the Timely Accumulation of Protein Phosphatase 1γ on Anaphase Chromosomes

“…As an exemplification, we have already identified another enzyme, an acetyltransferase, which targets to mitotic chromosomes and is dependent on Ki67 for this targeting. 4 To date, many proteins have been reported to localize to the surface of mitotic chromosomes (39,40,42). It will be interesting to examine which of these proteins and their biological activities are localized in a manner dependent on Ki67.…”

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

“…The results indicate that the identified phosphoproteins spanned a range of abundance from over a million to less than 500 copies per cell (supplemental Table S1). More than 40% of the phosphoproteins identified in this study are core components of mitotic chromatin (42). Detailed analysis of the kinetochore, a macromolecular structure larger than 1 MDa that assemble on the centromere of mitotic chromosomes, indicated that the gentle conditions used for the purification of mitotic chromatin (Experimental Procedures) preserved even labile protein-protein interactions (43,44) and thus retained even loosely associated proteins in the chromatin fraction.…”

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

“…A similar assembly pathway is also observed in an in vitro assembly system where the process of histone deposition and chromatin contraction occurs within 30 s (16,17). Regardless of this apparent rapid compaction, it takes much longer for new chromatin to become indistinguishable from the bulk chromatin in vivo (9, 13).Recent systematic studies revealed that mature chromatin adopts a complex molecular structure containing a large variety of binding factors that go way beyond a simple aggregate of DNA and histones (11,12,18,19). This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation.…”

“…Recent systematic studies revealed that mature chromatin adopts a complex molecular structure containing a large variety of binding factors that go way beyond a simple aggregate of DNA and histones (11,12,18,19). This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation.…”