alpha5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings.

The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat alpha5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense lambda expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71. It bears 50. 0% and 46.3% sequence identity with rat and yeast proteasome subunit alpha5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the alpha5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa. Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T. brucei 20 S proteasome) antiserum. In view of the documented self-assembly of the archaeon proteasome alpha subunit into double heptamer rings and the spontaneous assembly of the two alpha subunits from the 20 S proteasome of Rhodococcus erythropolis, the self-assembly of the T. brucei alpha subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals.