title: Quantitative analysis of cohesin complex stoichiometry and SMC3 modification-dependent protein interactions.
abstract: Cohesin is a protein complex that plays an essential role in pairing replicated sister chromatids during cell division. The vertebrate cohesin complex consists of four core components including structure maintenance of chromosomes proteins SMC1 and SMC3, RAD21, and SA2/SA1. Extensive research suggests that cohesin traps the sister chromatids by a V-shaped SMC1/SMC3 heterodimer bound to the RAD21 protein that closes the ring. Accordingly, the single "ring" model proposes that two sister chromatids are trapped in a single ring that is composed of one molecule each of the 4 subunits. However, evidence also exists for alternative models. The hand-cuff model suggests that each sister chromatid is trapped individually by two rings that are joined through the shared SA1/SA2 subunit. We report here the determination of cohesin subunit stoichiometry of endogenous cohesin complex by quantitative mass spectrometry. Using qConCAT-based isotope labeling, we show that the cohesin core complex contains equimolar of the 4 core components, suggesting that each cohesin ring is closed by one SA1/SA2 molecule. Furthermore, we applied this strategy to quantify post-translational modification-dependent cohesin interactions. We demonstrate that quantitative mass spectrometry is a powerful tool for measuring stoichiometry of endogenous protein core complex.
authors.affiliation: Center for Molecular Discovery, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Ding
authors.forename: Yehua
authors.initials: Y
authors.lastname: Li
authors.forename: Beom-Jun
authors.initials: BJ
authors.lastname: Kim
authors.forename: Anna
authors.initials: A
authors.lastname: Malovannaya
authors.forename: Sung Yun
authors.initials: SY
authors.lastname: Jung
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D000097722:Cohesins