An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

Abstract

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein’s molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.

Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing

“…AP-MS resulted in fewer high-confidence interactions than BioID (Figure 2A), likely because BioID can also detect more transient interactions. Similar differences between these two approaches have been observed previously for various proteins (Lambert et al, 2015;Liu et al, 2018). Most of the shared hits from the AP-MS and BioID ( Figure 2B) include already established interactions of actin in chromatin remodeling complexes, including SWI/SNF (Zhao et al, 1998) (ARP4, BRG1 and BAF170) and SRCAP/TIP60 (Cai et al, 2005;Ikura et al, 2000) (ARP4, EP400, YEATS4, DMAP1).…”

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

“…We identified a total of 143 biotinylated proteins in Ykt6-WT expressing cells enriched over background ( Supplementary Table 2). In general, BioID captures weak and transient protein-protein interactions and proximate proteins (Liu et al, 2018). Interestingly, we did not identify other SNAREs in the BioID approach.…”

Nudt8 is a novel CoA diphosphohydrolase that resides in the mitochondria

“…This substrate specificity, in turn, may be relevant to the physiological function of Nudt8. Using independent spatially restricted enzyme tagging-based approaches, two studies have recently identified human NUDT8 as a resident of the inner membrane/matrix in HEK293T cells [42,43], which suggests that Nudt8 may regulate the matrix CoA pool. Interestingly, significant activation by Mn 2+ has also been observed for other mitochondrial Nudix hydrolases including mouse Nudt13, an enzyme that degrades NADP(H), and AtNudt15, a CoA diphosphohydrolase in Arabidopsis thaliana that, however, was not tested against acetyl-CoA [37,38].…”

“…While the localization of Nudt8 places it in a position to modulate mitochondrial metabolism, the precise physiological role of this enzyme will depend on the submitochondrial CoA pool that it can actually access. Using independent spatially restricted enzyme tagging-based approaches, two studies have recently identified human NUDT8 as a resident of the inner membrane/matrix in HEK293T cells [42,43], which suggests that Nudt8 may regulate the matrix CoA pool. Such localization would also potentially place Nudt8 in the same compartment as CoA synthase (Coasy), the last enzyme in the CoA biosynthetic pathway [44][45][46].…”