Identifying Protein-Protein Associations at the Nuclear Envelope with BioID

Abstract

Summary The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which these NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein association as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID-fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.

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

“…Some of the top cellular component terms included nuclear periphery (GO:0034399), nuclear lamina (GO:0005652), and nuclear matrix (GO:0016363) ( Figure 2C). To further examine if our data was consistent with previous studies we benchmarked our data on four published studies of nuclear envelope interactomes [21][22][23]27,28,36,37] : ( 1 ) Bar et al combined a novel antibody based proximity labeling strategy and a meta-analysis of many lamin A and B interactome studies that included methods such as BioID, yeast two hybrid, and co-immunoprecipitation. .…”

“…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] . Not surprisingly, the majority of Lap2β protein-protein interactions uncovered in this study were previously identified as part of the laminome.…”

“…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.…”

“…To better understand these proximal protein compartments, we have leveraged the BioID system to study the "micro-proteome" of both LADs and the nuclear lamina using a multi-pronged approach. First, we have generated a chimeric protein comprising a modified promiscuous biotin ligase, BirA*, fused to the nucleoplasmic N-terminus of LAP2β to analyze the interactome of this important protein of the INM/lamina ( Figure 1A) [22,23] . Second, we have taken a novel approach combining the specificity of a DamID-based system to label LADs in live cells, the m6A-tracer system, and coupled this with BioID to characterize proteins proximal to LADs ( Figure 1B, C) [16] .…”