Identifying true protein complex constituents in interaction proteomics: The example of the DMXL2 protein complex

Abstract

A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions.

Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

“…Loss of the v-ATPase subunit isoform ATP6V0A1 (v0a1) in zebrafish microglia [27] or the Drosophila ortholog vha100-1 in photoreceptors [28] produces a subcellular defect similar to our results with rbc3a -MO1 injection: that is, aggregation of early endosome/phagosomes that do not mature but still become acidified. Additionally, co-IP experiments with mouse Rbc3a show specific interactions with V0a1 but not other V0a subunit isoforms [23]. Therefore we tested requirements for V0a1 in vesicle trafficking and acidification during early NC migration.…”

“…Rabconnectin-3a (Rbc3a) is a large, ∼325 kDa protein, highly conserved in multicellular organisms, which associates with its obligate binding partner, Rbc3b, and subunits of the v-ATPase complex [22],[23]. Studies in Drosophila embryos and murine cell culture have shown that Rbc3a and Rbc3b are required for proper lysosomal acidification and regulation of the Notch signaling pathway [24],[25].…”

Rare copy number variations affecting the synaptic gene DMXL2 in neurodevelopmental disorders

“…Dmxl2 +/− mice also demonstrate neuroanatomical differences in the corpus callosum [68]. At least four of ten experimentally proven protein interactors of DMXL2 [69] are encoded by established or candidate genes for NDDs: CYFIP2 [70, 71], DYNC1H1 [72, 73], MATR3 [74], and NCKAP1 [5, 75, 76]. Both CYFIP2 and NCKAP1 shape the formation of dendritic spines via the WAVE actin-remodeling complex [77, 78].…”

The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3β/WDR7 in neuroendocrine cells

“…1, E–H ). Because V-ATPase subunits were also enriched in CAPS1 immunoprecipitates relative to control and because the Rbcn3 complex and V-ATPase were reported to interact (18, 19, 55), we suggest that CAPS1 is part of a Rbcn3/V-ATPase complex that could regulate DCV function.…”