Structure and functional analyses of the 26S proteasome subunits from plants - Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated pr oteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a col lection encoding 12 out of 18 known eukaryotic regulatory particle (RP) sub units, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the correspond ing orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed w ith the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates . RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degr adation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN1 0 is not essential for in vivo functions. Instead, a domain near the N-term inus is required. We have begun to exploit the moss Physcomitrella patens a s a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 Delta strains which grow normally, Physcomitrella rpn10 Delta strains are developmentally arrested, being unable to initiate gamet ophorogenesis. Further analysis of these mutants revealed that RPN10 is lik ely required for a developmental program triggered by plant hormones.