INACTIVATION OF THE 20S PROTEASOME IN MYCOBACTERIUM-SMEGMATIS

The 20S proteasome is an essential component of the cytosolic protein turnover apparatus of eukaryotic cells. In higher eukaryotes, the 20S proteasome is responsible for most cytosolic protein turnover and also generates peptides for subsequent presentation by the MHC class I pat hway. Structurally, the eukaryotic 20S proteasome is extremely complex , being composed of 14 different subunits. Proteasomes with simplified subunit composition have been identified in certain eubacteria and ar chaebacteria but, in each case, the proteasome-containing organism is recalcitrant to further molecular genetic analyses. As a result, no in vivo characterization of a simplified eubacterial or archaebacterial proteasome has been reported. We have shown that the genetically tract able eubacterium Mycobacterium smegmatis contains a 20S proteasome, al lowing the first in vivo characterization of a simplified 20S proteaso me. We use a positive/negative selection scheme to inactivate the gene s encoding 20S proteasome subunits and demonstrate that, in contrast t o eukaryotic cells, M. smegmatis cells lacking intact proteasome genes are viable and phenotypically indistinguishable from congenic strains containing proteasomes. Implications for the evolution of the protein turnover apparatus are discussed.