Table 1.
Overview of methods and conditions used to identify UFM1 substrates and interacting proteins.
| Method | Constructs and Experimental Conditions | Substrates or Interacting Proteins | Reference |
|---|---|---|---|
| Affinity purification | Strep-Tag UFM1 overexpressed in insulin-producing MIN6 cells | UBA5, UFC1, UFL1, DDRGK1 | [33] |
| GST-UFM1 | |||
| Flag-His-UFM1 | ASC1 | [34] | |
| Flag-UFL1 and DDRGK1 | p53 | [58] | |
| His6-UFM1ΔSC | RPL26 and RPL26L1 | [35,36] | |
| Flag-UFM1 (WT and ΔC3) in UFSP2 CRISPR knockout cells and mass spec | |||
| Flag-DDRGK1 | ANT3 1, NUP160 1, and SF3B1 1 (DDRGK1 associated proteins) | [76] | |
| BirAOPT-UFM1-UFC1 vector | 20 UFM1 substrates/interactors found incl. MRE11. Only CYB5R3 and PSMB5 were validated | [125] | |
| StrepII-tagged UFM1 | RPL26, RPS3 1, RPS20 1, RPL10 1 and several UFM1 interactors | [81] | |
| Yeast two-hybrid screening | Originally GATE16 was discovered as a UBA5 interactor, and later identification of UFM1 by AP-MS | [15] | |
| Yeast two hybrid and PTM reconstitution in E. coli | pDEST32 bait and prey vectors; pYESS-PIP and pYESSPTM vectors | 16 potential substrates or interactors; only DDRGK1, MT1M1, and TSC22D3 1 were validated | [126] |
| Co-immunoprecipitation and microscopy experiments | His-UFM1 pulldown under denaturing conditions after induction of DNA damage followed by MS/MS | Histone H4 and three other potential substrates (ZNF281 1, ZIC2 1, and CAPNS1 1) | [39] |
| Colocalization of UFL1 and y-H2AX upon irradiation; co-immunoprecipitation of HA-UFL1 revealed interaction with MRE11 | MRE11 | [40] | |
| Protein microarray and ELISA | GST-NCAM140 (cytoplasmic domain) | UFC1 interaction with the cytoplasmic domain of NCAM 140 | [91] |
| Protein microarray | Profiling of Ubl modification of about 9000 proteins before and after release from mitotic arrest | Identification of 202 UFM1 targets (transmembrane transporters, ion channels, cytokine transporters); hit validation required | [55] |
| Peptide array | UBA5 peptides and GST-LC3 and GST-GABARAP | Mapping of LIR/UFIM motif in the C-terminal domain of UBA5 | [19] |
| Peptide-competition coupled affinity proteomics screen | IP-MS screen under ER stress conditions using a synthetic ATG8-interacting motif peptide with higher affinity than ATG8 | C53 (or CDK5RAP3) was revealed to bind ATG8 | [68] |
| CRISPR screens | CRISPR knockout screens (CRISPRi) | p62 (SQSTM), DDRGK1, RPN1 | [38,59] |
| CRISPR-mediated HA-tagging of endogenous proteins (hATG8) | ACSL3 | [56] | |
| Chromatin Co-immunoprecipitation (ChIP) | Endogenous and overexpressed XBP-1s (Flag-XBP-1s) | XBP-1s binds to the promoter region of UFM1 | [54] |
| Bioinformatic analysis | Analysis of proteins with UFM1 binding motif [19] | STK38 (NDR1) binding to UFMylated Histone H4 in complex with KAP-1 1 and SUV39H1 | [104] |
1 ANT3 (ADP/ATP translocase 3); NUP160 (nuclear pore complex protein Nup160); SF3B (splicing factor 3B subunit 6); RPS3 (40S ribosomal protein S3); RPS20 (40S ribosomal protein S20); RPL10 (60S ribosomal protein L10); MT1M (Metallothionein-1M); TSC22D3 (TSC22 domain family protein 3); ZNF281 (zinc finger protein 281); ZIC2 (zinc finger protein ZIC2); CAPNS1 (calpain small subunit 1); KAP-1 (also known as TRIM28 or transcription intermediary factor 1 beta).