BioID Identification of Lamin-Associated Proteins

Abstract

The nuclear lamina is primarily composed of type-V intermediate filaments, the A- and B-type lamins, which give mechanical support to the nuclear envelope, contribute to heterochromatin organization and regulate a myriad of nuclear processes. Over a dozen human diseases, collectively named laminopathies, are associated with mutations in the genes that encode the nuclear lamins. Although the etiology of the laminopathies is well understood, the molecular mechanisms by which they lead to disease remain unclear. Also poorly understood are the mechanisms by which the lamins contribute to a variety of nuclear and cellular functions. The identification of proteins associated with the lamins is likely to provide insight into these fundamental mechanisms. In recent years a unique method for identifying protein-protein and proximity-based interactions has emerged, called BioID (proximity-dependent biotin identification). BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry (MS). BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. The application of BioID is amenable to insoluble proteins such as lamins that are often refractory to study by other methods and is capable of identifying weak and/or transient interactions. In this review, we discuss the use of BioID as a powerful tool to help elucidate novel interacting partners of lamins.

The MyMOMA domain of MYO19 encodes for distinct Miro‐dependent and Miro‐independent mechanisms of interaction with mitochondrial membranes

“…Once they reached approximately 80% confluency, they were grown in the presence of 50 μM biotin overnight (minimum of 8 hr). Biotinylated proteins were purified as previously described (Mehus, Anderson, & Roux, ). Briefly, cells were lifted from the plate using 0.25% trypsin EDTA, and washed with PBS via centrifugation (700 g , 4 °C, 10 min).…”

“…Biotinylated proteins were purified as previously described (Mehus, Anderson, & Roux, 2016 (1:5,000, ThermoFisher) for 40 min, and then washed in PBS three times for 5 min. Blots were then exposed to Clarity ECL (Bio-Rad) for 5 min, and visualized using the BioRad ChemiDoc MP imager.…”

Analysis of K-Ras Interactions by Biotin Ligase Tagging

“…We have modified the promiscuous biotin ligase tagging system (85)(86)(87)(88) to analyze the protein interaction networks of the G12D, WT, S17N, and C185S/G12D K-Ras proteins. As shown, our chimeric BirA*Ras proteins were expressed as expected, localized as predicted, and their localization patterns matched the patterns of the proteins they biotinylated (Figures 1-4).…”

Mapping the micro-proteome of the nuclear lamina and lamin associated domains

“…Previous studies have identified proteins enriched at the nuclear lamina (the "laminome") through multiple methods, including BioID [21][22][23][24][25] . Here we use BioID with BioSITe (BioID coupled with biotinylation site enrichment and analysis) to measure protein-protein interactions of the INM protein Lap2β (Figure 2) in MEFs and integrate these with the previous laminome findings to generate an enhanced INM/lamina proteome map [21][22][23]27,28,36,37] .…”

“…These coiled-coil domain proteins provide a structural scaffold at the INM, with the A-type lamins being shown to mediate LAD organization [12,[17][18][19] . Longstanding efforts have been undertaken to map and characterize the local proteome of the nuclear lamina of the INM using inner nuclear membrane preparations, co-immunoprecipitation and, more recently, BioID ( Bio tinylation Id entification), a method for detecting proximal protein interactions in living cells [21][22][23][24][25] . However, these efforts have exclusively focused on protein members of the INM/lamina as baits and have not measured the protein landscape of LADs themselves or the intersection of these proteome environments.…”