Proteomics

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

ABSTRACT: 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.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens  

PROVIDER: MSV000080759 | MassIVE | Wed Mar 29 09:59:00 BST 2017

SECONDARY ACCESSION(S): PXD006206

REPOSITORIES: MassIVE

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In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome by attaching a promiscuous biotin ligase ('BioID') to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic  ...[more]

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