Fig. 3: The deubiquitinase Otu2p protects TOCs from degradation during gametogenesis.

a Schematic domain structure of OTUB (protease C65) family deubiquitinases (DUBs). OTUB family members are distinguished by a UBA (ubiquitin-associated) domain in the catalytic OTU domain and lack N-terminal UBL (UBX-like) and C-terminal zf-C2H2 domains conserved among OTUD proteins. OTU2 encodes an 1166 or 1174 amino acid (aa) protein with a C-terminal OTU domain. Otu2 homologs include a unique loop (orange triangle, 55 aa-long in C. reinhardtii) within the UBA domain. The predicted catalytic triad (vertical red lines; D901, C904, and H1151 in Otu2_C. reinhardtii) is conserved in OTUB and OTUD. Divergent OTUB family proteins in monocotyledonous plants (Otu1L) and mammals (Otub2), but lack the N-terminal UID domain known to interact with E2 enzymes. A ubiquitin-interacting motif (UIM) is found in Otu2 homologs. b In vitro deubiquitinase activity assay for the otubain domain of Otu2. The deubiquitinase activity against the tetra-ubiquitin substrates in Lys48-linked and Lys63-linked forms was examined by SDS-PAGE and immunoblot analysis using an anti-ubiquitin antibody. The in vitro deubiquitinase reaction was incubated for 30 min at 37 °C with 50 ng proteins. The C-terminal otubain domains of Otu2p, Otu2pC904S, and Otu2m proteins as well as the full-length Arabidopsis Otu1 (AtOtu1) proteins were produced in E. coli and purified by N-terminal 6x His-tag. The assay was repeated more than three times with technical duplicates.