Proteomics

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

ABSTRACT: To capture the spatial organization of the dynein/dynactin motor, we attached a promiscuous biotin ligase (BioID) to 6 distinct activators of the dynein machinery motile activity. BioID experiments were performed with stable HEK293 cell lines expressing full length BICD1, BICD2, HOOK1, HOOK3, NIN and NINL with BioID tags at their N-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.

INSTRUMENT(S): LTQ

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

SUBMITTER: Laurence Florens  

PROVIDER: MSV000080807 | MassIVE | Thu Mar 30 10:13:00 BST 2017

SECONDARY ACCESSION(S): PXD006220

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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