MassIVE MSV000080759

Partial Public PXD006206

MudPIT analyses of the human proteins biotinylated by proximity to dynein/dynactin subunits fused to biotin ligase (BioID)

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Description

We identified the human dynein proteome by attaching a promiscuous biotin ligase (BioID) to 7 distinct components of the dynein machinery, including a subunit of its essential cofactor dynactin. BioID experiments were performed with stable HEK293 cell lines expressing dynein (IC1, IC2, LIC1, LIC2, RB, TcTex-1) and dynactin (p62) subunits tagged with BioID-3X FLAG. BioID-only transfected cells were used as negative controls. For BioID experiments, cells were lysed in the presence of detergents to disrupt the dynein/dynactin complex, allowing the identification of proteins that were proximal to the tagged subunit prior to cell lysis. After purification of biotinylated proteins on streptavidin-conjugated beads, the eluates were precipitated with TCA. After washing with acetone, the protein mixtures were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by MudPIT. We performed BioID purification followed by MudPIT in quadruplicate and used a label-free quantitative proteomics approach to calculate the enrichment of each identified protein relative to BioID control replicates. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Cytoplasmic dynein-1 ; dynactin ; activator ; promiscuous biotin ligase

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Redwine WB, DeSantis ME, Hollyer I, Htet ZM, Tran PT, Swanson SK, Florens L, Washburn MP, Reck-Peterson SL.
The human cytoplasmic dynein interactome reveals novel activators of motility.
Elife. Epub 2017 Jul 18.

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