BIOCHEMICAL PURIFICATION OF DISTINCT PROTEASOME SUBSETS

Proteasomes are intricate cellular proteases that are able to degrade many protein and peptide substrates in vitro. These particles are stru cturally complex; they are assembled from at least 14 small molecular mass polypeptide subunits to form mature 20S proteasomes. Recently, we demonstrated that proteasome subsets may be discriminated by serologi cal criteria, and have found that subtle differences in the subunit co mposition of proteasomes can alter their cleavage specificity. Proteas ome structural complexity is further enhanced when some proteasomes as sociate with additional proteins to form a 26S ATP- and ubiquitin-depe ndent protease. Herein we confirm the existence of distinct cellular f orms of proteasomes, and show that they differ in their hydrophobic ch aracteristics. We have reproducibly purified, using solely biochemical techniques, distinct proteasome subsets similar to the serologically defined LMP2(+) and LMP2(-) proteasomes. These proteasome subsets diff er in their expression of at least three polypeptides, and both lack s everal additional polypeptides as compared to the serologically define d LMP2(+) and LMP2(-) proteasomes. Finally, we demonstrate that these structurally unique proteasomes differ in their capacity to cleave a d efined panel of fluorogenic peptide substrates. It appears that mammal ian cells might recruit and modify proteasomes to perform distinct cel lular tasks.