BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

Abstract

The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF ␤-TrCP1 and SCF ␤-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF ␤-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF ␤-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ␤-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ␤-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. Molecular & Cellular

Landscape of nuclear transport receptor cargo specificity

“…Methodologically, our analysis complements previous work that relied on alternative approaches. The major advantage of in situ proximity ligation is that it is done in living cells and thus preserves all subcellular structures, protein concentrations, and regulatory networks (Hung et al , 2014; Coyaud et al , 2015), while several previous studies relied on protein extraction or permeabilized cells. Nevertheless, cross‐validation against such previous data showed that similar set of cargos are identified (Kimura et al , 2017).…”

“…The AP was performed as previously described (Coyaud et al , 2015). Instead of a protease inhibitor mixture, 1 mg/ml aprotinin and 0.5 mg/ml leupeptin was used.…”

A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function

“…Hence, it is possible that HWS contributes to the miRNA pathway through a protein not yet described in this contextthat the interaction with an already known factor is weak or transient, as described for other F-box proteins (Earley et al, 2010;Coyaud et al, 2015)-or that the concentration of the interactor in entire leaves is low and therefore not detectable by our approach. Although we are confident that the F-box domain is important for HWS's function in the miRNA context, its mode of action and interaction(s) with miRNA factors remain to be determined.…”

Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

“…If a larger labeling radius is desired, for example to capture more constituents within a large protein complex, it can be increased by insertion of flexible linkers between the bait and the ligase [23]. BioID also captures protein associations over a period of time and independent of stable interactions, making it an effective approach in identifying novel substrates of proteins with dynamic and transient protein associations such as the E3 ubiquitin ligase, β-TrCP1 [24]. …”