MassIVE MSV000080808

Partial Public PXD006221

MudPIT analyses of the human proteins biotinylated by proximity to biotin ligase (BioID) fused to the C-terminus of activators of the dynein/dynactin motor

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Description

To understand how a single motor achieves cargo specificity, we attached a promiscuous biotin ligase (BioID) to 6 distinct activators of the dynein machinery motile activity, including two new activators, ninein and ninein-like. BioID experiments were performed with stable HEK293 cell lines expressing full length BICD1, BICD2, HOOK1, HOOK3, NIN and NINL with BioID tags at their C-termini. 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 activator 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 (Promega) 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 ; coiled-coil ; cargo specificity

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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