Functional Analysis of Arabidopsis BRIZ Proteins

Eukaryotes have adopted a multi‐enzymatic Ub/26S proteasome system to regulate protein degradation, which consists E1, E2 and E3. E3, the ubiquitin ligase, is often the key component for substrate specificity and a major type of E3 ligase uses a RING domain as the E2 docking site. Previous analysis has shown that there are more than 400 RING‐domain containing proteins in the Arabidopsis predicted proteome and some have been shown to catalyze polyubiquitination in vitro . However, most of them do not have defined in vivo functions. My project focuses on functional analysis of two related Arabidopsis RING‐domain containing proteins, BRIZ1 and 2. Both of these proteins have multiple predicted domains; a BRAP2, RING, ZnF‐UBP, and coiled‐coil domain from the N‐terminus to C‐terminus. Despite their sequence similarity and shared predicted domains, no homozygous T‐DNA insertion lines can be isolated from insertions in either loci, indicating that they are not functionally redundant. Heterozygous plants from insertions in BRIZ1 or BRIZ2 produce 3:1 viable:inviable seed. Homozygous mutant lines are recovered from both the BRIZ1 and 2 T‐DNA insertion lines after introduction of a plasmid containing coding sequences, indicating that disruption of BRIZ1 or 2 is responsible for the lethality. These observations suggest that both proteins together might be essential for proper germination through complex formation. BRIZ1 and BRIZ2 interacted after synthesis in in vitro transcription and translation reactions. In addition, GST‐tagged BRIZ1 and 6His‐tagged BRIZ2 preferentially interacted. These observations strongly suggest that hetero‐dimer formation is favored over homo‐dimer formation.