Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans

Abstract

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His₁₀ tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

Keywords: APEX2; Caenorhabditis elegans; HRP; IMAC; TurboID; biotinylated carboxylase depletion; proximity-dependent protein labeling; synapse.

Figure 1

Optimizing TurboID-mediated proximity labeling in Caenorhabditis elegans.A, schematic overview of the TurboID protocol, including depletion of His₁₀-tagged endogenously biotinylated carboxylases. B, Western blot showing the efficiency of carboxylase depletion using a Ni–NTA resin and extracts from C. elegans expressing MCCC-1::His₁₀; PCCA-1::His₁₀; POD-2::His₁₀; PYC-1::His₁₀ (abbreviated as MP3-His). C, Western blot analysis of extracts from animals expressing elks-1::TbID::mNG in an MP3 background. One round of Ni–NTA depletion efficiently removes His₁₀-tagged MP3. FT: flow through after carboxylase depletion; His1 and His2: eluted His-tagged carboxylases captured by Ni–NTA resin after 1 (His1) or 2 (His2) rounds of His depletion; POI: protein of interest; input: whole lysate without carboxylase depletion; control: rab-3p::mNG; mp3-His; Free TbID: rab-3p::TbID::mNG; mp3-His; emp3: elks-1::TbID::mNG; mp3-His. Red asterisk: TurboID-mNeongreen; black asterisks: endogenously biotinylated carboxylases. MCCC-1: methylcrotonoyl coenzyme A carboxylase 1; Ni–NTA: nickel–nitrilotriacetic acid; PCCA-1: propionyl coenzyme A carboxylase alpha subunit 1.

Figure 2

Interactome analysis of the presynaptic active zone protein ELKS-1 by comparing carboxylase depleted versus undepleted samples.A, significantly enriched proteins in ELKS-1 undepleted samples compared with wildtype (log₂ ≥1.5-fold change threshold, adjusted p ≤ 0.05). As expected, many synaptic proteins are highly enriched. B and C, significantly enriched proteins when data obtained from analysis of 50% (B) or 5% (C) of depleted samples are compared with 5% of undepleted ELKS-1 samples (log₂ ≥1.5-fold change threshold, adjusted p ≤ 0.05). D, Venn diagram highlighting number of significantly enriched proteins between samples. See also Fig. S3A. E, table showing log₂ fold enrichment and adjusted p value of synaptically annotated proteins. ED: elks-1::TbID::mNG; mp3-His₁₀ carboxylase depleted; WT: mp3-His₁₀; E: elks-1::TbID::mNG carboxylase undepleted.

Figure 3

Previously uncharacterized proteins enriched in depleted samples.A, significantly enriched proteins in the ELKS-1 depleted samples (ED-50%) compared with undepleted ones (E-5%). Please note that the volcano plot used in this panel is the same volcano plot used for Figure 2B. B and C, mean spectral counts (B) and log₂ fold enrichment (C) of previously uncharacterized proteins R148.3 and F59C12.3 obtained via mass spectrometry. D, confocal microscopy images of transgenic C. elegans expressing R148.3::mNG or F59C12.3::mNG translational fusion in the DA9 motor neuron. elks-1::mSc, used as a control to mark DA9 synapses, confirms these proteins are synaptic. The cartoon below the micrographs illustrates the anatomy of the DA9 neuron, highlighting the dendrite, cell body, and the presynaptic region, which is enclosed in a dashed box. ELKS-1::mScarlet is represented as red dots in the presynaptic region. The dashed box corresponds to the area imaged in the micrographs above. The scale bars represent 5 μm.