Abstract:
:The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a "promiscuous" biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is "frozen." This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.
journal_name
Mol Biol Celljournal_title
Molecular biology of the cellauthors
Remnant L,Booth DG,Vargiu G,Spanos C,Kerr ARW,Earnshaw WCdoi
10.1091/mbc.E18-12-0799subject
Has Abstractpub_date
2019-05-15 00:00:00pages
1314-1325issue
11eissn
1059-1524issn
1939-4586journal_volume
30pub_type
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