Fig 2: Displacement of the long RARS from the multi-synthetase complex induces ATE1 redistribution into the cytosol. (A) Sample fractionation showing the relative levels of ATE1 in the total extract (total), the cytosol and the nuclear fraction, probed with tubulin (a largely cytosolic marker) and histone H3 (a nuclear marker) to confirm the fractionation efficiency. (B) Chart and representative immunoblots of ATE1 levels in the cytosol of wild-type (parental) and dLZ cells. (C) Chart and representative immunoblots of total ATE1 levels in wild-type and dLZ cells. Error bars represent the SEM; n = 3 biological replicates. ** p < 0.01, Welch’s t-test.

Fig 3: ATE1 can interact with the long and short RARS, but overexpression of RARS does not facilitate arginylation. (A) Schematic representation of the RARS constructs used for immunoprecipitation and overexpression. Start codons (AUG) were individually mutated to AUC to abolish the HA tag from the expression construct and ensure the start of translation either on both sites (WT) or on the upstream or downstream AUG individually (for the long and short RARS, respectively). (B) Representative immunoblots of the immunoprecipitation steps from HEK293T cells transfected with either the short or long RARS as indicated on the top and visualized with RARS and ATE1 antibodies. FT, flow-through; P, pulldown. The top and bottom panels show the same blot at different exposures to visualize the typical immunoprecipitation steps (top), as well as the minor levels of ATE1 in the precipitates (bottom, marked with asterisks and arrows on the right). The positions of the short and long RARS are indicated in the top panel as S and L, respectively. IgG, immunoglobulin heavy chain. The pulldowns were repeated at least 3 independent times with similar results. (C) Chart and representative immunoblots of arginylation sensor quantification in HEK293T cells transfected with different RARS isoforms as indicated. U, untransfected control. High and low exposure images of the same gel are shown to enable a comparison of the levels of endogenous RARS with the transfected RARS isoforms. Error bars represent SD; n = 3 biological replicates. ns, not significant, one-way ANOVA.

Fig 4: Interplay between ATE1 and RARS. In wild-type cells (left), ATE1 exists in a balance with free and MSC-bound RARS. Knockout of the leucine zipper (right) that scaffolds the long RARS to the MSC perturbs this balance by reducing the nuclear MSC and ATE1 localization, resulting in an increase in cytoplasmic ATE1 activity. Cytosolic and nuclear environments are denoted with white and blue backgrounds, respectively.

Fig 5: The ATE1–RARS interaction does not depend on tRNA. Quantification (top) and representative immunoblots (bottom) of purified ATE1 and RARS pulldowns in the presence and absence of tRNA, RNase A and cell extract, as indicated in the table on the bottom under each lane and bar (+ represents addition of each component in each line). Error bars represent SD; n = 3 independent pulldowns. ns, not significant, one-way ANOVA.