A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

J Cell Biol. 2012 Mar 19;196(6):801-10. doi: 10.1083/jcb.201112098. Epub 2012 Mar 12.

Abstract

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Biotin / metabolism*
  • Biotinylation
  • Carbon-Nitrogen Ligases / genetics
  • Carbon-Nitrogen Ligases / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • HEK293 Cells
  • Humans
  • Laminin / genetics
  • Laminin / metabolism*
  • Protein Interaction Mapping / methods*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*

Substances

  • Escherichia coli Proteins
  • Laminin
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • laminin A
  • Biotin
  • Carbon-Nitrogen Ligases
  • birA protein, E coli