Compiled list of the Urm1-interacting proteins in <i>Drosophila</i> embryos, larvae and adults, identified by mass spectrometry.

<p>Compiled list of the Urm1-interacting proteins in <i>Drosophila</i> embryos, larvae and adults, identified by mass spectrometry.</p

<i>In vivo</i> confirmation of the binding of Urm1 to a panel of target proteins identified by mass spectrometry.

<p>Confirmation of a physical interaction between Urm1 and five of the newly identified candidate targets of urmylation. Using the UAS/GAL4-system, Flag-Urm1 was either expressed alone, or co-expressed separately with HA-Ciao1, HA-MsrA/Eip71CD, GFP-GILT1 or GFP-Crammer, respectively, under control of the Actin5C-GAL5 driver. In protein lysates from the resulting flies, the interaction between Urm1 and the candidate target proteins were subsequently analyzed by immunoprecipitation, performed in in the presence of NEM. By immunoblotting Flag-Urm1 immunoprecipitates with anti-Jafrac1, anti-HA or anti-GFP antibodies, an interaction could thereby be verified between Flag-Urm1 and endogenous <i>Drosophila</i> Jafrac1 (A), HA-tagged Ciao1 (B), HA-tagged MsrA/Eip71CD (C), GFP-tagged GILT1 (D) and GFP-tagged Crammer (E). When comparing the molecular weights of the candidate Urm1 target proteins following immunoprecipitation, a size shift of ~15 kDa could be observed for Jafrac1 (A), HA-Ciao1 (B), HA- MsrA/Eip71CD (C) and GFP-GILT1 (D) as compared with protein lysate controls, which is in agreement with the covalent conjugation of one Flag-Urm1 moiety (e.g. target protein urmylation). In contrast, GFP-Crammer displayed the same molecular weight in both Flag-Urm1 immunoprecipitates and crude fly lysates, depicting a non-covalent mode of interaction, which is sensitive to denaturing conditions (E). Input represents 30 μg of the total lysate of the indicated genotypes.</p

Identification of Urm1-binding proteins by mass spectrometry.

<p><b>A, C, E.</b> Heat maps depicting a correlation analysis of the mass spectrometry results obtained from the two control <i>Actin5C>w</i>^<i>1118</i> replicate reads (Control replicate 1 and 2), versus the two Flag-Urm1 rescued <i>Urm1</i>^<i>n123</i> <i>Drosophila</i> replicate reads (Rescue replicate 1 and 2) for embryos (A), larvae (C) and adults (E), respectively. For all developmental stages, the two control and rescue replicates show high similarity, respectively, with a clear distinction between the controls versus the rescue samples. The color key code represents the Pearson correlation coefficient of the two replicates, where 1 depicts 100% similarity between the two replicates and 0 depicts no correlation between the samples. <b>B, D, F.</b> Volcano plots illustrating the magnitude of differential distribution (log2 fold-change) of the signal intensity between the Flag-Urm1 rescue and the control samples, together with the adjusted p-value for embryonic (B), larval (D) and adult (F) samples, respectively. Red dots depict peptides that displayed a log2 fold-change of less than 1.3 and a high adjusted p-value, when comparing the rescue and control samples. Blue dots represent peptides that did not show any significant difference between the rescue and the control samples, with a lower adjusted p-value. Green dots pinpoint peptides that demonstrated a low adjusted p-value and were enriched in the rescue samples to a minimum of 1.3 log2 fold-change, as compared with the control samples. The green dots represent the peptides that were finally singled out as Urm1-interacting proteins, with the cut-off marked by a dashed yellow line.</p

Functional characterization of the newly identified candidate targets of Urm1 conjugation in <i>Drosophila</i>.

<p><b>A.</b> Subcellular distribution of the proteins identified as Urm1-binding partners by mass spectrometry, depicting an accumulation of candidate targets of urmylation in the cytoplasmic compartment and/or associated with distinct membrane-bound organelles. <b>B.</b> Venn diagram illustrating the amount of unique versus shared Urm1-interacting proteins in the developmental stages investigated; embryos, larvae and adults. <b>C.</b> Functional clustering of the Urm1-associated proteins established using the DAVID database, suggesting that Urm1 most likely displays its most important functions in oxidation-reduction processes and tRNA modification. Enrichment scores of >3.0 were considered as meaningful. <b>D.</b> A functional classification of the Urm1-binding proteins based on gene ontology (GO) classification suggests that Urm1 may be involved in multiple different biological processes.</p

Protein-protein interaction analysis by STRING clusters Urm1 conjugation targets in multiple distinctive functional networks.

<p>STRING analysis of the Urm1-intercting partners identified by mass spectrometry, depicting functional networks where Urm1 may be involved in embryos (A), larvae (B) and adult flies (C). During all developmental stages, Urm1 appears to be associated with networks of proteins that regulate oxidation-reduction processes, tRNA modification, immune responses, mRNA processing, translation and protein folding, as well as cytoskeletal dynamics. In embryos, Urm1 is additionally linked to a protein network which is involved in chromatin remodeling.</p

Strategy for identification of Urm1 conjugation targets in <i>Drosophila</i> embryos, larvae and adults <i>in vivo</i>.

<p><b>A.</b> Schematic representation of the rationale for identifying Urm1-binding proteins and thereby candidate targets of urmylation during three critical stages of <i>Drosophila</i> development; embryogenesis, late larval stages and adulthood. Shortly, Flag-Urm1 associated proteins were enriched by immunoprecipitation with Flag M2 magnetic beads, which subsequently were subjected to on-bead trypsin digestion, followed by mass spectrometry and identification by standard bioinformatics analysis. <b>B.</b> Western blot illustrating the distribution of candidate Urm1 targets in embryos (left panel), larvae (middle panel) and adults (right panel), respectively. Urm1-interacting proteins were captured in the presence of NEM by Flag M2 immunoprecipitation, using magnetic beads, resolved under denaturing conditions by SDS-PAGE and detected by anti-Urm1 antibodies (* depicts endoge^◆nous Urm1 expressed in control <i>Actin5C>w</i>^<i>1118</i> samples and indicates the unconjugated Flag-Urm1 fusion protein). Input represents 30 μg of the total lysate of the indicated genotypes.</p