Roux et al. report a new way to screen for protein–protein interactions in mammalian cells.
Existing methods for probing protein interactions have their limitations. A yeast two-hybrid screen, for example, involves expressing proteins in a non-native cell type that may not fold or modify them correctly. Biochemical “pull-down” approaches, on the other hand, are limited by protein solubility and can miss weak or transient interactions. Roux et al. developed a new method called proximity-dependent biotin identification, or BioID, which relies on a promiscuous mutant of the bacterial biotin ligase BirA that biotinylates nearby primary amines, such as lysine residues in neighboring proteins.
The researchers fused the mutant ligase to human lamin-A, an insoluble component of the protein meshwork that underlies the inner nuclear membrane. When the fusion protein was expressed in cells, it localized to the nuclear envelope and biotinylated nearby proteins, which could be purified on biotin-binding beads and identified by mass spectrometry. This approach detected many nuclear membrane proteins and nuclear pore complex components known to associate with lamin-A. It also identified a previously uncharacterized protein that the authors localized to the nuclear envelope and named soluble lamina-associated protein of 75 kD, or SLAP75.
BioID identifies both a protein's binding partners and its near neighbors, says senior author Kyle Roux. In addition to using the technique with different target proteins, Roux wants to examine how disease-linked mutations in lamin-A alter the protein's association profile.