SILAC-based discovery of a broad range of proteins that can be posttranslationally glutamylated or glycylated

The posttranslational modifications (PTMs) glutamylation and glycylation are primarily associated with tubulins and microtubule regulation, whereas their broader functional scope is largely underexplored. It is known that both PTMs also occur on other proteins, yet only a few non-tubulin substrates have been identified so far. To gain a broad view of the potential roles of these two PTMs, we established a stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomics approach to identify substrates in an unbiased manner. Expressing glutamylating and glycylating enzymes in cell lines strongly increased the levels of these PTMs, from which modified proteins were purified with PTM-specific antibodies and identified with quantitative proteomics. We identified more than 100 putative substrates for both PTMs, including proteins involved in nucleocytoplasmic shuttling, RNA and chromatin-binding, and translation regulation, out of which we validated a representative subset. Our work provides a reliable resource for substrates of glutamylation and glycylation that opens the opportunity to functionally explore the roles of these understudied PTMs in a variety of cellular processes.