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Beyond Immunoprecipitation: Exploring New Interaction Spaces with Proximity Biotinylation Tess Branon,† Shuo Han,‡ and Alice Ting*,†,‡ †
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California 94305, United States
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richer interaction maps. For example, for the 10 shared baits, >40% of the interactors detected by IP were also detected using BioID; these shared interactions, however, represented only 21% of the interactors identified by BioID. BioID was also used to map the interaction space of the non-sense-mediated mRNA decay (NMD) complex.4 BioID proximity labeling identified a putative novel interactor of the NMD complex, elongation factor EIF4A2, that was missed with IP. EIF4A2 was then validated as a true interactor through proximity ligation assays, and further assays showed that EIF4A2 associates with the NMD complex during translation initiation, but not during elongation. The transient nature of the interaction may be the reason it was missed by conventional IP. Another advantage that proximity biotinylation presents over IP is that the former can give a higher level of enrichment of true interactors. Because the biotin probe covalently tags proteins, and the biotin−streptavidin interaction used for enrichment of labeled proteins is extremely strong, very stringent and extensive washes can be employed during purification to remove proteins that are nonspecifically bound. In contrast, washes for IP must be gentle so that they do not dissociate noncovalent protein−protein interactions; this results in higher background from nonspecific proteins. Indeed, in a side-by-side comparison by Chu et al. in this issue, the Ku70 and Ku80 interaction partners of the microprotein modulator of retroviral infection (MRI) were enriched 10-fold with APEX, but only ∼2-fold with IP.1 A last advantage to note for proximity biotinylation is that it can potentially produce more specific hits than IP, i.e., decrease the detection of false positives. Contrary to the traditional view that IP is highly specific, a number of studies have shown that some of the interaction partners detected using IP can result from nonspecific interactions that occur after cell lysis. For example, a study investigating the interactions of ribonucleoprotein complexes definitively showed that some of the IPenriched interactors had resulted from rearrangement postlysis and did not recapitulate the in vivo state.5 Upon cell lysis, biologically irrelevant interactions can form when proteins, which could be partially unfolded by the detergents used for lysis, come into contact with proteins with which they would not interact under physiological conditions. This can be particularly troublesome for proteins that are intrinsically unstructured or unfolded, such as some microproteins. Nonspecific interactions detected by IP were indeed problematic in the study by Chu et al., in which microproteins were found by IP to interact with both housekeeping and heat shock
ecently, a critical step toward the elucidation of the function of a largely uncharacterized class of proteins known as microproteins has been made.1 Microproteins are peptides and small proteins with fewer than 150 amino acids that are encoded by small open reading frames (smORFs). While hundreds to thousands of smORFs and their microprotein products have been identified by novel genomic and proteomic techniques, only a handful have been characterized to reveal their biological functions. Because all characterized microproteins seem to participate in microprotein−protein interactions to regulate biology, Chu et al. in this issue used both classical immunoprecipitation and APEX-mediated proximity biotinylation to map microprotein−protein interactions in an attempt to elucidate novel functions and pathways.1 APEX, a peroxidase that catalyzes proximitydependent biotinylation in living cells, proved to be advantageous to immunoprecipitation in this study and permitted the discovery of novel protein interaction partners of the previously uncharacterized microprotein C11orf98. Immunoprecipitation (IP) is a widely used technique for investigating protein−protein interactions (PPIs) from living cells. IP is straightforward to perform with antibodies against endogenous proteins or against epitope tags on recombinant proteins and has permitted countless biological discoveries. Whereas IP enriches proteins on the basis of their biochemical affinity for the bait protein of interest, proximity biotinylation is an alternative approach that is based on through-space biotinylation of proteins 1−10 nm from the APEX-fused bait. While these techniques are complementary to each other, proximity labeling possesses some notable advantages over traditional IP. One advantage of proximity biotinylation is that it can be used to detect weak or transient interactions that are missed by IP. For example, APEX was used in a recent study2 to identify and spatiotemporally resolve interaction partners of G-proteincoupled receptors as they progress through their dynamic signaling and trafficking pathways in response to ligand-induced activation. APEX proximity labeling identified the GPCR interaction partner arrestin3, while IP missed this interaction unless cross-linking was employed to stabilize and trap the interaction. The capability to detect weak and transient interactions is shared with other proximity labeling methods such as BioID, which has been used to map complex interaction networks in structures such as the centrosome−cilium interface.3 In this study, IP was performed alongside BioID proximity labeling for a subset of the bait proteins used. In this side-by-side comparison, many more functionally relevant interactors were detected using proximity labeling than using IP, leading to © XXXX American Chemical Society
Received: May 15, 2017
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DOI: 10.1021/acs.biochem.7b00466 Biochemistry XXXX, XXX, XXX−XXX
Viewpoint
Biochemistry proteins.1 The artifactual interactions were likely formed during cell lysis for IP. Because proximity biotinylation is performed in living cells before lysis, and because stringent, denaturing washes are used to separate APEX-biotinylated proteins from nonspecific binders after lysis, detection of false-positive PPIs that occurred postlysis can be circumvented. As such, the nonspecific microprotein interactors that were detected using IP, such as tubulin and HSPA9, were not detected in a parallel experiment using proximity labeling instead. In summary, proximity labeling approaches, including APEX and BioID, offer key advantages over traditional IP for protein− protein interaction analysis and discovery: capture of transient interaction partners (i.e., fewer false negatives), greater enrichment of true interactors, and higher capture specificity (i.e., fewer false positives). Because of the advantages and opportunities that proximity labeling can provide, this complementary approach is worth considering for any PPI mapping study.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Chu, Q., Rathore, A., Diedrich, J. K., Donaldson, C. J., Yates, J. R., and Saghatelian, A. (2017) Identification of microprotein-protein interactions via APEX tagging. Biochemistry, DOI: 10.1021/acs.biochem.7b00265. (2) Lobingier, B. T., Hüttenhain, R., Eichel, K., Miller, K. B., Ting, A. Y., von Zastrow, M., and Krogan, N. J. (2017) An approach to spatiotemporally resolve protein interaction networks in living cells. Cell 169, 350−360. (3) Gupta, G. D., Coyaud, É., Gonçalves, J., Mojarad, B. A., Liu, Y., Wu, Q., Gheiratmand, L., Comartin, D., Tkach, J. M., Cheung, S. W., Bashkurov, M., Hasegan, M., Knight, J. D., Lin, Z. Y., Schueler, M., Hildebrandt, F., Moffat, J., Gingras, A. C., Raught, B., and Pelletier, L. (2015) A dynamic protein interaction landscape of the human centrosome-cilium interface. Cell 163, 1484−1499. (4) Schweingruber, C., Soffientini, P., Ruepp, M. C., Bachi, A., and Mühlemann, O. (2016) Identifcation of interactions in the NMD complex using proximity-dependent biotinylation (BioID). PLoS One 11, e0150239. (5) Mili, S., and Steitz, J. A. (2004) Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10, 1692−1694.
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DOI: 10.1021/acs.biochem.7b00466 Biochemistry XXXX, XXX, XXX−XXX
