Characterization of the 20S proteasome from the actinomycete Frankia

Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic ass ociation with the root systems of a variety of non-leguminous plants, denom inated actinorhizal plants. Information on the biology of proteolysis in Fr ankia is almost non-existent as it is extremely difficult to grow this orga nism. We have purified 20S proteasomes from Frankia strain ACN14a/ts-r. It is composed of one alpha-subunit and one beta-subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme di splayed a chymotrypsin-like activity against synthetic substrates and was s ensitive to lactacystin, a specific proteasome inhibitor. Analysis of the s tructural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coel icolor and showed that the beta-subunit is encoded with a 52-amino-acid pro peptide that is cleaved off in the course of the assembly. We report also f or the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembl ed and proteolytically active.